KR20170099992A - Modified APRIL binding antibody - Google Patents

Abstract

This disclosure relates to an APRIL-binding antibody that binds to an epitope of the same human APRIL as an antibody having an antigen binding site of hAPRIL.01A. The antibodies of the present disclosure include a specific selection of the framework sequences of the V _H and V _L domains and have unexpected characteristics compared to hAPRIL.01A. The present disclosure further relates to compositions comprising the antibodies of the invention and to medical and diagnostic uses of the antibodies and compositions.

Description

Modified APRIL binding antibody

The present invention relates to isolated antibodies comprising fragments thereof, to a human APRIL, to polynucleotides encoding such antibodies and to host cells producing such antibodies. Antibodies can be used to treat cancer and to treat conditions that can benefit from immune cell proliferation and inhibition of such immune cell proliferation, such as autoimmune diseases, inflammatory diseases, or diseases associated with immunoglobulin excess production . In addition, antibodies may be used as diagnostic tools and as in vitro preparations for the inhibition of immune cell proliferation and / or survival.

APRIL is expressed as a type II transmembrane protein, but unlike most other TNF family members, it is processed as a secreted protein and cleaved at the Golgi, where it is cleaved by a purine-converting enzyme to release a soluble active form (Lopez-Fraga et al. , 2001, EMBO Rep 2: 945-51). APRIL is assembled as non-covalently linked homo-trimers with similar structural homology to protein folding for many other TNF family ligands (Wallweber et al., 2004, Mol Biol 343, 283-90). APRIL binds to two TNF receptors, B-cell matured antigens (BCMA) and membrane permeant activators and calcium modulators and cyclophilin ligand interactant (TACI) (Kimberley et al., 2009, J Cell Physiol . 218 1): 1-8]. In addition, APRIL has recently been shown to bind heparan sulfate proteoglycan (HSPG) (Hendriks et al., 2005, Cell Death Differ 12, 637-48). APRIL plays a role in B cell signaling and has been shown to drive both proliferation and survival of human and murine B cells in vitro (Kimberley et al., 2009, J Cell Physiol . 218 (1): 1- 8].

APRIL is mostly expressed by a subset of immune cells, such as monocytes, macrophages, dendritic cells, neutrophils, B-cells, and T-cells, many of which also express BAFF. In addition, APRIL can be expressed by non-immune cells, such as osteoclasts, epithelial cells and various tumor tissues (Kimberley et al., 2009, J Cell Physiol . 218 (1): 1-8 ]). In fact, APRIL was originally identified on the basis of its expression in cancer cells (Hahne et al., 1998, J Exp Med 188, 1185-90). High expression levels of APRIL mRNA have been identified in one panel of tumor cell lines as well as in human primary tumors, such as colon, and lymphocytic carcinoma.

95 In a retrospective study of chronic lymphocytic leukemia (CLL), CLL patients exhibited increased levels of APRIL in serum, which was associated with poor prognosis and poor prognosis in patients with disease progression and overall patient survival, high APRIL serum levels (Planelles et al., 2007, Haematologica 92, 1284-5). Similarly, APRIL has been shown to be expressed in Hodgkin lymphoma, non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM) (Kimberley et al., 2009, J Cell Physiol . 218 ): 1-8]. Retrospective studies in DLBCL patients (NHL) showed high APRIL expression in cancerous lesions associated with poor survival (Schwaller et al., 2007, Blood 109, 331-8). Recently, APRIL serum levels in serum from patients suffering from colorectal cancer have been shown to have positive diagnostic values (Ding et al., 2013, Clin. Biochemistry, http://dx.doi.Org/10.1016/j. Clin .

Due to its role in B cell biology, APRIL also plays a role in many autoimmune diseases. Increased serum levels of APRIL have been reported in many SLE patients (Koyama et al., 2005, Ann Rheum Dis 64, 1065-7). Retrospective analysis revealed that APRIL serum levels have a tendency to be associated with anti-dsDNA antibody titers. Significantly increased levels of APRIL were also detected in patients with inflammatory arthritis compared to those suffering from non-inflammatory arthritis, for example, osteoarthritis (Stohl et al., 2006, Endocr Metab Immune Disord Drug Targets 6 , 351-8; Tan et al., 2003, Arthritis Rheum 48, 982-92).

Several studies have focused on the presence of APRIL in the serum of patients suffering from a wide range of systemic immune-based rheumatic diseases (currently including Sjogren's syndrome, Reiter's syndrome, psoriatic arthritis, multiple myelitis and Ankylosing Spondylitis) (Jonsson et al., 1986, Scand J Rheumatol Suppl 61, 166-9; Roschke et al., 2002, J Immunol 169, 4314), which has been shown to have an increased level of APRIL -21). In addition, increased APRIL serum levels were detected in serum from patients suffering from atopic dermatitis (Matsushita et al., 2007, Exp. Dermatology 17, 197-202). In addition, serum APRIL levels rise in sepsis and predict death in severely ill patients (Roderburg et al., J. Critical Care, 2013, http://dx.doi.org/10.1016/j.jcrc.2012.11. 007 ). In addition, APRIL serum levels were found to increase in patients suffering from IgA nephropathy (McCarthy et al., 2011, J. Clin. Invest . 121 (10): 3991-4002).

Finally, increased APRIL expression was also linked to multiple sclerosis (MS). APRIL expression was found to be increased in astrocytes of MS patients compared with normal controls. (Deshayes et al., 2004, Oncogene 23, 3005-12; Roth et al., 2001, Cell Death Differ 8, 403-10), which is associated with APRIL expression in sera from patients with glioblastoma and glioblastoma.

APRIL plays an important role in the survival and proliferative capacity of several B-cell malignant tumors, and potentially also some solid tumors. APRIL also appears to play an important role in inflammatory diseases or autoimmunity. Thus, the strategy of antagonizing APRIL has a therapeutic purpose in many of these diseases. Indeed, clinical studies are currently underway to target APRIL to TACI-Fc (Atacicept) for the treatment of some autoimmune diseases. However, TACI-Fc also targets BAFF, a factor associated with normal B-cell maintenance. Antibodies against APRIL are described in WO9614328, WO2001 / 60397, WO2002 / 94192, WO9912965, WO2001 / 196528, WO9900518 and WO2010 / 100056. WO2010 / 100056 describes an antibody that specifically targets APRIL. The antibody of WO2010 / 100056 completely blocks binding of APRIL to TACI and at least partially blocks binding to BCMA. The antibody hAPRIL.01A completely blocks binding to both BCMA and TACI. The hAPRIL.01A antibody inhibited B-cell proliferation, survival and antigen-specific immunoglobulin secretion in vitro and in vivo (Guadagnoli et al., 2011, Blood 117 (25): 6856-65). In addition, hAPRIL.01A inhibited the proliferation and survival of representative malignant cells of human CLL and MM disease in vitro and in vivo (Guadagnoli et al., 2011, Blood 117 (25): 6856-65; Lascano et al , 2013, Blood 122 (24): 3960-3; Tai et al., 2014, ASH poster 2098). Finally, hAPRIL.01A inhibited the secretion of antigen-specific IgA (Guadagnoli et al., 2011, Blood 117 (25): 6856-65). In view of these unique binding characteristics, such murine antibodies have unique pharmacological utility. However, there are also certain disadvantages to the pharmacological utility of such antibodies in human medicines in terms of their murine origin. The present invention therefore aims to provide a modified hAPRIL.01A antibody more suitable for use in human pharmaceuticals.

The present invention provides hAPRIL.01A analogs comprising specific substitutions of the framework regions of the V _H and V _L domains. wherein the framework region of the V _H domain of hAPRIL.01A at the antigen binding site of hAPRIL.01A replaces the framework region from the V _H amino acid sequence selected from SEQ ID NO: 12, 14, 16 or 18 and the V _{L of} hAPRIL.01A It has surprisingly been found that when the framework region of the domain replaces the framework region of the V _L amino acid sequence selected from SEQ ID NO: 30, a functional hAPRIL.01A analog is obtained. This indicates that research by the inventors of the present invention has shown that only a limited set of alternative V _H and V _L framework sequences from human origin can support the proper binding of hAPRIL.01A CDRs to human APRIL, It is surprising in the sense that it has shown that it can cause .01A analogs. In addition, the inventors of the present invention have found that further improvement in hAPRIL.01 analogs can be obtained by introducing double-substituted R67K in combination with certain specific amino acid substitutions, particularly amino acid substitutions R72S and / or V68A in the selected V _H amino acid sequence .

The invention according to the first aspect thus provides an antibody having an antigen binding site of hAPRIL.01A, for example an APRIL-binding antibody which binds to an epitope of the same human APRIL as the monoclonal antibody hAPRIL.01A described in WO2010 / 100056 Wherein the human APRIL-binding antibody comprises a plurality of antigen binding sites, including V _H and V _L domains, wherein the framework sequence of the V _H domain at the antigen binding site comprises SEQ ID NO: 12, 14, 16 or 18 , Preferably at least 70% sequence similarity to the framework sequence of the V _H amino acid sequence selected from SEQ ID NO: 14 or 18, most preferably SEQ ID NO: 18, and the framework sequence of the V _L domain comprises V 0.0 > 70% < / RTI > sequence similarity to the framework sequence of the _L amino acid sequence.

A further aspect of the present invention relates to a polynucleotide, an expression unit comprising a plurality of polynucleotides, and an expression unit and / or a plurality of polynucleotides, which encode the variable region of the heavy and light chains of the antibody of the invention, ≪ / RTI >

A further aspect of the invention is a method of producing an antibody of the invention,

a) culturing the host cell of the present invention in a culture medium under conditions in which a plurality of polynucleotides are expressed, thereby producing a polypeptide comprising a heavy chain variable region; And

b) recovering the polypeptide from the host cell or culture medium. Compositions comprising an antibody of the invention in combination with a pharmaceutically acceptable carrier or diluent and optionally a plurality of other active compounds are subjects of further aspects of the invention.

The therapeutic and diagnostic uses of the antibodies of the invention are another aspect of the invention.

A brief description of the sequence

The sequences shown in the sequence listing relate to the V _H and V _L domains and the amino acid sequences and encoding DNA sequences of the heavy and light chains from which the framework sequences can be used in the antibodies according to the present invention. In addition, both the V _H and V _L domains of hAPRIL.01A and the amino acid sequences of the heavy and light chain CDRs are present. According to a particular embodiment of the invention, the CDRs of hAPRIL.01A are used in the antibodies of the invention. Table 1 below is related to the sequence numbers for each of these sequences.

SEQ ID NOS: 11 to 52 relate to engineered immunoglobulin VH, VL, heavy or light chain sequences as indicated.

Figure 1 shows the results of targeting APRIL to hAPRIL.01A or analog 14_1G.15 in vivo for T cell-independent B cell responses. Transgenic mice (APRIL-Tg) were challenged with 250 μg NP-Ficoll (0 day) and treated with hAPRIL.01A or 14_1G.15 on days -1 and 3. PBS and wild-type mice (WT) were used as negative control. Immunoglobulin titers (IgA1 (panel a) and IgA2 (panel b), IgG (panel c) and IgM (panel d)) were measured by ELISA. 14_1G.15 inhibited the T-cell independent immune response to NP-phycols, which is more effective than its hAPRIL.01A analog.

The present invention therefore relates to an antibody that binds to an epitope of the same human APRIL as an antibody comprising an antibody analog, e. G. An antibody fragment, having an antigen binding site of hAPRIL.01A. The antibody hAPRIL.01A is described in WO2010 / 100056 along with the sequences of the V _H and V _L domains and CDRs. The inventors of the present invention have confirmed that there is a limited possibility of substituting the mouse framework region of the V _H and V _L domains of hAPRIL.01A as human substitutes. Further selected alternative framework sequences result in alternative anti-human APRIL antibodies with unexpected characteristics. The selected framework sequences reveal their particular characteristics within the context of binding to the human APRIL epitope for hAPRIL.01A and are therefore considered to have broad applicability in this context. Accordingly, the present invention is directed to any antibody (including fragments and / or derivatives and / or analogs) that binds to the same epitope as hAPRIL.01A, including framework sequences selected from its V _H and V _L domains . In certain embodiments, such antibodies will comprise alternative CDRs that differ from the CDRs of hAPRIL.01A. However, according to another embodiment, the antibody will comprise similar or even identical CDRs to those of hAPRIL.01A. According to a particular embodiment, the antibody comprising the V _H domains CDR1, CDR2 and CDR3 of hAPRIL.01A and variants of the V _L domains CDR1, CDR2 and CDR3 or any of the above sequences is an epitope of the same human APRIL as the monoclonal antibody hAPRIL.01A ≪ / RTI > This is particularly the case when binding to human APRIL with a K _D of about 100 nM or less or blocking the binding of human APRIL to human BCMA and TACI with an IC ₅₀ of about 100 nM or less. Preferred antibodies in the present invention are human monoclonal antibodies directed against human APRIL at a concentration of about 100 nM or less, such as 100 to 0.001 nM, such as 100 to 0.010, 100 to 0.050, 100 to 0.100, 100 to 0.150, 100 to 0.200, 100 to 0.300, 100 to 0.350, 100 to 0.400, 100 to 0.450, 90 to 0.500, 80 to 0.550, 70 to 0.600, 60 to 0.650, 50 to 0.700, 40 to 0.750, 30 to 0.800, 20 to 0.850, from 0.900 to 1.0 to 0.950nM or combines with K _D values in the selected interval. Those skilled in the art will appreciate that lower values such as values of 50, 20, 10, 1.00 nM or less are preferred for K _D. Antibodies with K _D values within these intervals are suitable for clinical applications (see, for example, Presta, et al., 2001, Thromb. Haemost. 85: 379-389); [Yang, et al., 2001 , Crit. Rev. Oncol. Hematol. 38: 17-23); [Carnahan, et al., 2003, Clin. Cancer Res. (Suppl.) 9: 3982s-3990s). Antibody affinity may be determined using standard assays known to those skilled in the art, for example, as exemplified in the experimental section.

The antibodies of the present invention may be used in an amount of about 100 nM or less, such as 100 to 0.001 nM, such as 100 to 0.010, 100 to 0.050, 100 to 0.100, 100 to 0.150, 100 to 0.200, 100 to 0.250, 100 to 0.300, 100 to 0.400, 100 to 0.450, 90 to 0.500, 80 to 0.550, 70 to 0.600, 60 to 0.650, 50 to 0.700, 40 to 0.750, 30 to 0.800, 20 to 0.850, 10 to 0.900, It is further preferred that the binding of human APRIL to human BCMA and TACI be blocked with an IC ₅₀ value in the interval selected from 0.950 nM. The skilled person will appreciate that lower values such as values of 50, 20, 10, 1.00 nM or less are desirable for IC ₅₀ .

Binding of the antibody to the same epitope as hAPRIL.01A can be achieved by the methods described in Example 2 or 5 of WO2010 / 100056 or by using the antibodies of the invention and hAPRIL < RTI ID = 0.0 > 0.0 > APRIL < / RTI > of a reference antibody having an antigen binding site of .01A. The antigen binding site of hAPRIL.01A is defined by the V _H and V _L domains as being present in SEQ ID NOS: 3 and 4. Thus, any antibody, including antibody fragments, including V _H and V _L domains designated SEQ ID NOS: 3 and 4, may be used to assess binding to epitopes of the same human APRIL as hAPRIL.01A Can be used as an antibody. The antibody hAPRIL.01A described in WO2010 / 100056 is an example of an antibody having an antigen binding site of hAPRIL.01A and is a suitable reference antibody in the context of the present invention. However, antibody fragments, for example, Fab, F (ab) ₂ or Fv fragments derived from hAPRIL.01A can also be used as reference antibodies. Based on the DNA sequences of SEQ ID NOs: 1 and 2 and the amino acid sequences of SEQ ID NOs: 3 and 4, the skilled artisan will be able to construct and produce such antibody fragments derived from the hAPRIL.01A roll. Based on the provided sequence, the skilled artisan will also be able to determine the binding of the heavy chain variable region amino acid sequence of SEQ ID NO: 3 (encoded by SEQ ID NO: 1) to the IgGl constant region (mouse or different species, preferably from a mouse) The hAPRIL.01A analog may be prepared for use as a reference antibody by binding of the light chain variable region amino acid sequence of SEQ ID NO: 4 (encoded by SEQ ID NO: 2) to a mouse or mouse, or from a different species, preferably from a mouse will be.

When assessed in this way, antibody binding to an epitope of human APRIL identical to hAPRIL.01A is less than or equal to about 100 nM, such as 100 to 0.001 nM, such as 100 to 0.010, 100 to 0.050, 100 to 0.100, 100 to 0.200, 100 to 0.250, 100 to 0.300, 100 to 0.350, 100 to 0.400, 100 to 0.450, 90 to 0.500, 80 to 0.550, 70 to 0.600, 60 to 0.650, 50 to 0.700, Can bind to a reference antibody having a hAPRIL.01A antigen binding site for human APRIL with an IC ₅₀ in the interval selected from the range of from about 0.750, 30 to 0.800, 20 to 0.850, 10 to 0.900 or 1.0 to 0.950 nM. The skilled artisan will appreciate that low values are desirable for values of IC ₅₀ , e.g., 50, 20, 10, 1.00 nM or less.

An antibody of the invention may thus have one or more of the following characteristics:

(i) binds to human APRIL with a K _D of about 100 nM or less;

(ii) blocking binding of human APRIL to human BCMA and human TACI with an IC ₅₀ of about 100 nM or less;

(iii) block binding of hAPRIL.01A to human APRIL with an IC ₅₀ of about 100 nM or less.

These features can be combined in the following combinations: (i) or (ii) or (iii); (i) and (ii); (i) and (iii); (ii) and (iii); (i) and (ii) and (iii).

The framework sequences of the V _H domain of the antigen binding site of the antibody according to the invention are selected such that they have at least 70% sequence similarity to the framework sequence of the V _H amino acid sequence selected from SEQ ID NOs: 12, 14, 16 or 18. According to a preferred embodiment, the framework sequences of the V _H domain of the antibody are selected so that they have at least 70% sequence similarity to the framework sequence of the V _H amino acid sequence selected from SEQ ID NO: 14 or 18, most preferably SEQ ID NO: 18. The framework sequences of the V _L domain at said antigen binding site of the antibody are selected such that they have at least 70% sequence similarity to the framework sequence of the V _L amino acid sequence selected from SEQ ID NO:

The V _H amino acid sequence selected from SEQ ID NOs: 12, 14, 16, or 18 and the V _L amino acid sequence selected from SEQ ID NO: 30 include both framework sequences and CDR sequences. The CDR sequences introduced into these V _H and V _L amino acid sequences are those of hAPRIL.01A and include SEQ ID NO: 5 (hAPRIL.01A V _H CDR1), 6 (hAPRIL.01A V _H CDR2), 7 (hAPRIL.01A V _H CDR3), 8 (hAPRIL.01A V _L CDR1), 9 (hAPRIL.01A V _L CDR2), 10 (hAPRIL.01A V _L CDR3). However, as described above, the use of selected V _H and V _L framework sequences in the present invention is not limited to combinations with the specific CDRs of hAPRIL.01A. Thus, at least 70% sequence similarity according to a particular embodiment is not the complete V _H amino acid sequence selected from SEQ ID NOs: 12, 14, 16, 18, or the complete V _L amino acid sequence selected from SEQ ID NO: 30, It is considered to be related. The framework sequences for V _H amino acid sequences shown as SEQ ID NOs: 12, 14, 16, or 18 include portions of these sequences outside the V _H CDRs, i.e., SEQ ID NO: 5 (hAPRIL.01A V _H CDR1), 6 (hAPRIL.01A V _H CDR2), 7 (hAPRIL.01A V _H CDR3). The framework sequence for the V _L amino acid sequence shown in SEQ ID NO: 30 includes the portion of this sequence outside the V _L CDR, i.e., SEQ ID NO: 8 (hAPRIL.01A V _L CDR1), 9 (hAPRIL.01A V _L CDR2) (hAPRIL.01A V _L CDR3).

According to an alternative embodiment, at least 70% sequence similarity is considered to be the complete V _H amino acid sequence selected from SEQ ID NO: 12, 14, 16 or 18 and the complete V _L amino acid sequence selected from SEQ ID NO: 30.

At least 70% sequence similarity in the context of the present invention means at least 80%, such as at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 99% sequence similarity .

Those skilled in the art will appreciate that "sequence similarity" refers to the degree to which individual nucleotide or peptide sequences are similar. The degree of similarity between two sequences is based on the degree of conservatism and the degree of identity combined. The percentage of "sequence similarity" is the percentage of amino acids or nucleotides that have been changed identically or conservatively, ie, "sequence similarity" = (% sequence identity) + (% conservative change).

For purposes of the present invention, "conservative changes" and "identity" are considered to be a somewhat broader term "similarity. Thus, when the term sequence "similarity" is used, it includes sequence "identity" and "conservative variation. Conservative changes are discarded according to certain embodiments, and% sequence similarity indicates% sequence identity.

The term "sequence identity" is known to the skilled artisan. In order to measure the degree of sequence identity shared by two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., the gaps are aligned in an optimal alignment with a second amino acid or nucleic acid sequence May be introduced as a first amino acid or sequence of the nucleic acid sequence. This sorting can be performed while comparing sequences of full length. In general, alignment may be performed on shorter comparative lengths, for example, about 20, about 50, about 100 or more nucleic acids / bases or amino acids.

The amino acid residue or nucleotide is then compared at the corresponding amino acid position or nucleotide position. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is the same at that position. The degree of identity shared between sequences is typically expressed in terms of percent identity between sequences, and is a function of the number of identical positions shared by the same residue in the sequence (i.e.,% identity = number of identical residues at corresponding positions / Total number of positions x 100). Preferably, the two sequences being compared are the same or substantially the same length.

The percentage of "conservative changes" can be measured similar to the percentage of sequence identity. However, in this case, the change in the specific position of the amino acid or nucleotide sequence that appears to substantially preserve the functionality of the original residue is recorded as a change does not occur.

The relevant functional properties in the amino acid sequence are the physical-chemical properties of the amino acid. Conservative substitutions for amino acids in the polypeptides of the present invention may be selected from other members of the class to which the amino acid belongs. For example, amino acids belonging to the grouping of amino acids having a particular size or characteristic (e.g., charge, hydrophobicity and hydrophilicity) may be modified substantially to alter the activity of the protein, particularly in the region of the protein not directly associated with biological activity (Watson, et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Edition 1987)). For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, cryptopan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, substituting Arg for Lys to maintain a positive charge or vice versa; Substituting Asp for Glu to maintain negative charge or vice versa; Substituting Thr for Ser so that free-OH is maintained or vice versa; Substituting Asn for Gin to replace free -NH ₂ , or vice versa.

Exemplary conservative substitutions in the amino acid sequence of a CD70 binding peptide of the invention can be made according to the following description as follows:

The relevant functional properties in the nucleotide sequence are biological information that only a particular nucleotide is contained within the open reading frame of the sequence in terms of transcription and / or translation machinery. It is common knowledge that genetic codes have axial (or overlap) and that multiple codons can have the same degree of amino acid that they encode. For example, amino acid leucine in certain species is encoded by the UUA, UUG, CUU, CUC, CUA, CUG codon (or TTA, TTG, CTT, CTC, CTA, CTG in DNA) , UCC, UCU, AGU, AGC (or TCA, TCG, TCC, TCT, AGT, AGC in DNA). Nucleotide changes that do not alter translated information are considered conservative changes.

Skilled artisans will appreciate that several different computer programs using different mathematical algorithms are available for measuring the identity between sequences. For example, use may be made of a computer program using the Needleman and Wunsch algorithms (Needleman et al. (1970)). According to an embodiment, the computer program is a GAP program of Accelrys GCG software package (Accelrys Inc., Diego, USA). Substitution matrices that can be used include, for example, BLOSUM 62 matrix or PAM 250 having a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, Matrix. Skilled artisans will recognize that all these different parameters will yield slightly different results, but when using different algorithms, the overall percent identity of the two sequences is not significantly altered.

Percent identity between two nucleotide sequences according to embodiments is measured using the GAP program of the Axcelis GCG software package (Axcelis Inc, Diego, USA). The NWSgapdna CMP matrix and the gap weight of 40, 50, 60, 70, or 80 and the length weight of 1, 2, 3, 4, 5, or 6 are used.

In another embodiment, percent identity of two amino acid or nucleotide sequences is measured using the algorithm of E. Meyers and W. Miller (Meyers et al. (1989)), Using an ALIGN program (version 2.0) (sequence data from Genestream server IGH Montpellier France) using the PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4, , Available at http://xylian.igh.cnrs.fr/bin/align-guess.cgi ).

In the present invention, it is most preferable to use a BLAST (Basic Local Alignment Tool) to measure percent identity and / or similarity between nucleotides or amino acid sequences.

Queries using BLASTn, BLASTp, BLASTx, tBLASTn, and tBLASTx programs from Altschul et al. ncbi.nlm.nih.gov] via the online version of BLAST. Alternatively, a standalone version of BLAST that is also downloadable via the NCBI Internet site (eg, version 2.2.29 (released January 3, 2014) may be used. Preferably, the BLAST query is performed with the following parameters. To determine percent identity and / or similarity between amino acid sequences: algorithm: blastp; Word size: 3; Matrix scoring: BLOSUM62; Gap cost: existence: 11, extension: 1; Composition control: conditional composition scoring matrix control; Filter: off; Mask: Off. To determine percent identity and / or similarity between nucleotide sequences: algorithm: blastn; Word size: 11; Maximum match in query scope: 0; Match / Mismatch Score: 2, -3; Gap cost: existence: 5, extension: 2; Filter: low complexity domain; Mask: A mask for navigation only.

The percentage of "conservative changes" can be measured as a percentage of sequence identity with the aid of the indicated algorithm and computer program. Some computer programs, for example, BLASTp, indicate the number / percentage of positive (= similarity) and the number / percentage of identity. The percentage of conservative changes can be derived from this by subtracting the percent of identity from the percentage of positive / similarity (percent conservative change = percent similarity-percent identity).

It will be appreciated by those skilled in the art that the antibodies of the invention will comprise multiple antigen binding sites. The framework sequences of the amino acid sequences of the selected V _H and V _L domains are combined at the antigen binding site with the CDR sequences. The specific combinations of framework sequences from the V _H and V _L domains contemplated by the present invention are shown in Table 3 below, where "X" represents the position of the combination of the sketched V _H and V _L domains.

The combination of framework sequences from these V _H and V _L domains results in antibodies with functional binding to human APRIL. In addition, as further shown in the experimental section, in the tests performed by the inventors of the present invention, when combining the VH sequence of the present invention with the VL sequence (VL15) of SEQ ID NO: 30, only antibodies having APRIL binding functionality It should be noted that only one was obtained. In the tested combinations of selected VH sequences and other VL sequences (VL10 to VL14), the antibodies obtained did not have functional APRIL binding properties. A further surprising effect of the combination of VH and VL sequences used in accordance with the present invention is improved (thermal) stability compared to antibodies with hAPRIL.01A VH and VL sequences as shown in the experimental section.

A combination of a VH framework sequence of SEQ ID NO: 30 and a sequence derived from SEQ ID NO: 18 or a sequence derived therefrom such as a VH framework sequence of SEQ ID NOs: 32, 34, 36, 38, These preferred combinations are indicated by the underlined "X" in Table 3 . The most preferred combination of the VL framework sequence from SEQ ID NO: 30 and the VH framework sequence from SEQ ID NO: 40 is shown in Table 3 as the "X" in bold (and underlined) fonts. Surprisingly, it has been found that the combination of these VL and VH framework sequences results in antibodies with additional advantageous characteristics, including beneficial stability characteristics and / or improved binding to human APRIL targets.

According to a particular embodiment, at least one of CDR1, CDR2, CDR3 in the V _H domain is selected from the group consisting of SEQ ID NOs: 5, 6, 7, or any variant of the sequence, respectively. Preferably, in the V _H domain, CDR1, CDR2 and CDR3 are selected from SEQ ID NOS: 5, 6, 7, or any variant of the sequence, respectively. These V _H domain CDR sequences correspond to the V _H domain CDRs of hAPRIL.01A.

According to a particular embodiment, at least one of CDR1, CDR2, CDR3 in the V _L domain is selected from the group consisting of SEQ ID NOs: 8, 9, 10, or any variant of the sequence, respectively. Preferably, CDRl, CDR2 and CDR3 in the V _L domain are selected from SEQ ID NOS: 8, 9, 10, or any variant of the sequence, respectively. These V _L domain CDR sequences correspond to the V _L domain CDRs of hAPRIL.01A.

According to a particular embodiment, the V _H domains CDR1, CDR2 and CDR3 at the antigen binding site of the antibody are selected from SEQ ID NOS: 5, 6, 7, or any variant of the sequence, respectively, and the V _L domains CDR1, CDR2 and CDR3 SEQ ID NOS: 8, 9, 10, or any variant of the sequence.

The inventors of the present invention have surprisingly found that further improvement can be made with antibodies that combine the VH and VL framework sequences used in the present invention. Specifically, the substitution R72S in the VH amino acid sequence results in improved binding to human APRIL.

Substituted R67K-V68A further combined in the V _H amino acid sequence also results in improved binding to human APRIL. According to a particular embodiment, the invention therefore relates to an antibody wherein the amino acid is S at position 72 in the V _H amino acid sequence. The VH amino acid sequence of SEQ ID NOS: 32, 34, 36, 38, 40 is an example of such a VH amino acid sequence having an S residue at position 72. According to another embodiment, the present invention relates to an antibody wherein the amino acid is K at position 67 and the amino acid is A at position 68 in the V _H amino acid sequence. A combination of all three amino acid substitutions R72S, R67K and V68A is also envisioned within the present invention. Thus, in accordance with another embodiment, the present invention relates to an antibody wherein the amino acid is S at position 72, the amino acid is K at position 67, and the amino acid is A at position 68 in the V _H amino acid sequence.

Except for the V _H and V _L domains, the antibody may additionally comprise a domain, for example, a suitable number of C _H domains and a suitable number of C _L domains. The C _H and C _L domains may be of human origin. Such domains also include domains that provide antibodies with modified (or blocked) Fc regions to provide altered effector function. See, for example, U.S. Patent No. 5,624,821; WO 2003/086310; WO2005 / 120571; WO2006 / 0057702; Presta, 2006, Adv. Drug Delivery Rev. 58: 640-656; Vincent and Zurini, Biotechnol. J. , 2012, 7: 1444-50; [Kaneko and Niwa, Biodrugs , 2011, 25: 1-11]. Such modifications can be used to enhance or inhibit the various responses of the immune system, with the possible beneficial effects on diagnosis and treatment. Substitutes for the Fc region include amino acid changes (substitution, deletion and insertion), glycosylation or deglycosylation, and multiple Fc additions. It is preferred to use an Fc region that exhibits a reduced Fc effector function in accordance with certain embodiments. An antibody of the invention according to certain embodiments may also have an Fc region derived from human IgG4 and / or an Fc region with a N297Q glycosylation deficiency mutation. The C _L domain can be selected from human kappa or lambda constant domains. Preferably, the human kappa C _L domain is used.

According to a particular embodiment of the present invention there is provided an antibody comprising Fc and C _L domains wherein the V _H domain amino acid sequence is selected from the group consisting of SEQ ID NO: 42, 44, 46, 48, 52, preferably SEQ ID NO: 48 or 52, Most preferably a heavy chain amino acid sequence having at least 70% sequence similarity to the amino acid sequence selected from SEQ ID NO: 52, and the V _L domain amino acid sequence is a light chain amino acid sequence having at least 70% sequence similarity to the amino acid sequence selected from SEQ ID NO:

Specific combinations of these heavy and light chains contemplated by the present invention are shown in Table 4 below, wherein "X" represents the position of the spheric heavy and light chain combination.

The preferred combination is indicated by an underlined "X ". A more preferred combination is indicated by the "X" in bold (and underlined) fonts.

According to a further aspect, the invention relates to isolated polynucleotides encoding the V _H and / or V _L domains of an antibody according to the invention. The polynucleotide sequence encoding the V _H domain is preferably selected from the group consisting of SEQ ID NOS: 11, 13, 15, 17, 31, 39, 41, 43, 45, 47, 51, preferably SEQ ID NOS: 17, 31, 51, more preferably a polynucleotide sequence having at least 70% sequence similarity with the polynucleotide sequence selected from SEQ ID NO: 51. The polynucleotide sequence encoding the V _L domain is preferably a polynucleotide sequence having at least 70% sequence similarity with the polynucleotide sequence selected from SEQ ID NO: 29 or 49, preferably SEQ ID NO: 49.

The invention further relates to an expression unit comprising a plurality of expression vectors comprising a plurality of polynucleotides according to the invention under the control of suitable regulatory sequences, wherein the number of polynucleotides is selected from the group consisting of the V _H of the antibody according to the invention Domain and V _L domain. The expression unit can be designed such that the polynucleotide sequence coding for the V _H domain and the polynucleotide sequence coding for the V _L domain can be the same expression vector. Thus, the expression unit may comprise a single vector. Alternatively, polynucleotide sequence coding for the V _H domain and polynucleotide sequence coding for the V _L domain may be different expression vectors. In such an embodiment, the expression unit will comprise a plurality, e. G., Two expression vectors.

A further aspect of the invention relates to a host cell comprising a plurality of polynucleotides of the invention and / or an expression unit of the invention. The expression unit is preferably an expression unit comprising an expression vector comprising both polynucleotide sequence coding for the V _H domain and polynucleotide sequence coding for the V _L domain.

An antibody of the invention can be any one of the following:

A chimeric antibody or fragment thereof;

- humanized antibodies or fragments thereof; or

An antibody fragment selected from the group consisting of Fab, Fab ', Fab'-SH, Fv, scFv, F (ab') ₂ , bispecific mAbs and diabodies. Humanized antibodies comprising a plurality of antigen binding sites based on the CDRs of hAPRIL.01A are preferred. It can be noted that the framework regions selected in accordance with the present invention are derived from human origin and thus can be suitably used to obtain humanized antibodies, particularly when combined with constant regions from human origin.

According to a further aspect thereof, the present invention provides a method of producing an antibody of the present invention,

a) culturing a host cell comprising a plurality of polynucleotides of the present invention and / or an expression unit of the present invention under conditions such that the polynucleotide is expressed in a culture medium to produce a polypeptide comprising a light chain and a heavy chain variable region; And

b) recovering the polypeptide from the host cell or culture medium.

The present invention further relates to compositions comprising an antibody of the invention in combination with a pharmaceutically acceptable carrier or diluent. Such compositions may, in one embodiment, comprise one or more antibodies. In one embodiment, the composition comprises one or more other active compounds in addition to one or more antibodies of the invention. Such a combination composition can be used, for example, in combination therapy in the treatment of cancer. In this case, the antibody may be combined with one or more conventional anti-cancer drugs. Other additional active compounds may be used in other combination therapies. It is not obligatory to have more than two active compounds in the same composition in combination therapy. Thus, also part of the invention is the combination or subsequent use of an antibody and other active compounds, wherein the antibody and other active compounds are administered simultaneously or sequentially.

As is apparent from the above description, the antibodies of the invention may be used for therapeutic and diagnostic, and other non-therapeutic purposes. The invention thus further relates to methods of using antibodies for therapeutic and diagnostic and other non-therapeutic purposes.

In one embodiment, the therapy comprises inhibiting immune cell proliferation and / or immune cell survival. In yet another embodiment, the treatment is aimed at the treatment of cancer. In one embodiment, the therapy comprises treatment of an autoimmune disease. In one embodiment, the therapy comprises treatment of an inflammatory disease. In one embodiment, the therapy comprises the treatment of Ig secretion mediated diseases, particularly IgA secretion mediated diseases. The therapeutic use of the antibodies of the invention will be discussed in more detail below.

The antibodies of the present invention can be used in non-therapeutic applications, for example, in vitro or in vitro techniques, such as flow cytometry, e.g., Western blotting, enzyme linked immunosorbent assay (ELISA) It can be applied in immunohistochemistry.

therapy

In view of the fact that the antibody of the present invention binds to human APRIL analogous to hAPRIL.01A, the antibodies of the present invention are suitable for use in therapies similar to hAPRIL.01A in the experimental sector with the improvements discussed above. Thus, the antibodies of the invention are suitable for the treatment of conditions known or expected to be alleviated by blocking the interaction of BCAC and / or TACI with human APRIL. As is known in the art, blocking the interaction of BCMA and / or TACI with human APRIL inhibits immune cell proliferation and / or survival, and thus blocking such immune cell proliferation and / or survival is beneficial, For example, it is valuable for diseases mediated by inflammatory diseases, Ig secretion and / or autoimmune diseases. Blocking the interaction of BCMA and / or TACI with human APRIL may also be beneficial in the treatment of cancer.

The autoimmune conditions for which the antibodies of the present invention may be beneficial include, but are not limited to, multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease and other inflammatory bowel diseases such as ulcerative colitis, systemic lupus erythematosus (SLE) Autoimmune thrombocytopenic purpura, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, multiple myositis, autoimmune thrombocytopenia, autoimmune thrombocytopenia, autoimmune thrombocytopenia, MG, Hashimoto thyroiditis, , Autosomal recessive diabetes mellitus, autoimmune hepatitis, autoimmune hepatitis, autoimmune hepatitis, autoimmune hepatitis, autoimmune hepatitis, autoimmune hepatitis, autoimmune hepatitis, autoimmune hepatitis, autoimmune hepatitis, Autoimmune hemophilia, autoimmune lymphocytic proliferative syndrome (ALPS), autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphocytic proliferative syndrome, autoimmune grapevine Retinitis, retinitis, Guilin Barre syndrome, arteriosclerosis and Alzheimer's disease.

In addition, the antibodies of the invention may also be beneficial in the treatment of other related conditions, such as graft / transplant rejection or allergic conditions, where a reduction in immune response is beneficial.

The antibodies of the present invention may also be used for the treatment of other conditions which are beneficial in reducing the levels of IgA, IgG, IgM, including IgA1 or IgA2, such as Ig secretion, particularly IgA secretion, Ig overproduction, , May be beneficial in the treatment of conditions associated with IgA, IgG, IgM overproduction, particularly IgA overproduction, including IgA1 or IgA2, or Ig deposition, particularly IgA deposition. Examples of such conditions include, but are not limited to, other forms of IgA nephropathy and glomerulonephritis, pediatric adiposity, pemphigus disease, Henoch-Schlenz purpura, and other autoimmune diseases associated with Ig deposition.

The "condition" treatment in the present invention includes any therapeutic use including prophylactic and therapeutic use of anti-human APRIL antibodies. Thus, the term "condition" may refer to a physiological condition in a prophylactic environment that is a disease state but also does not alter the physiology to a deleterious state.

The cancer in the present invention can be used for the treatment and / or prevention of leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, myeloblastic leukemia cells, bone marrow mononuclear leukemia, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Neurogenic lymphoma, Burkitt's lymphoma and marginal B-cell lymphoma, intrathoracic lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, valdendrogmaglobulinemia, heavy chain disease, solid tumors, sarcoma and carcinoma, fibrosarcoma, The present invention also relates to a method of treating or preventing a disease selected from the group consisting of liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, cholesteatoma, vascular sarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, Pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, adenocarcinoma, sebaceous carcinoma, papillary adenocarcinoma, papillary adenocarcinoma, A tumor of the cervix, a tumor of the uterus, a tumor of the testis, a tumor of the lung, a small cell lung carcinoma, a non-small cell lung carcinoma, a small cell carcinoma, Neoplasm, neuroblastoma, retinoblastoma, coccygeal carcinoma, esophageal carcinoma, papillary carcinoma, papillary carcinoma, angioblastoma, neuroblastoma, meningioma, meningioma, melanoma, neuroblastoma, retinoblastoma, (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, head and neck cancer, head and neck cancer, Gastric cancer, intracavitary neoplasms, renal cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; Oral cancer (e.g., lips, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; But are not limited to, cancer of the respiratory system, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary cancer. Cancers of particular interest include cancer expressing APRIL, for example, malignant tumors derived from B-cells, lymphoid or colon or lung carcinoma or multiple myeloma (MM) or chronic lymphocytic leukemia (CLL) B cells to be.

For the treatment of any of the above-mentioned conditions, the antibodies of the present invention may be administered to a subject directly, alone or in combination with other therapeutic agents. Thus, in accordance with certain embodiments of the present invention, the antibody of the present invention among its uses and / or compositions may be used for the treatment of multiple myeloma (MM), myelodysplastic syndrome (MDS), valgentrogromaglobulinemia, B-CLL, diffuse large B cell A number of therapeutic agents used to treat lymphoma, non-Hodgkin's lymphoma and Wegener's granulomat, such as melphalan, vincristine, fludarabine, chlorambucil, vendamustine, etoposide, doxorubicin, cyclophosphamide, Can be combined with cisplatin. In addition, in the use and / or composition thereof, an antibody of the invention may be combined with a plurality of immunomodulators, such as corticosteroids (dexamethasone, prednisolone), thalidomide analogs (thalidomide, lenalidomide, have. Also, in the uses and / or compositions thereof, the antibodies of the invention may be combined with a number of targeted kinase inhibitors, such as, for example, ibrutinib, idurarris. In addition, in the use and / or composition thereof, the antibodies of the invention may be administered in combination with a number of antibody therapies targeting CD20, such as rituximab, opatumum, ovinotoumuim; Or antibody therapies targeting CD52, such as alemtuzumab; Or antibody therapies that target CD38, such as daratu mum; Or antibody therapies that target IL-6 or IL-6 receptors (e. G., Salilmate, tacyliumphem); Or antibody therapy for targeting CS-1 (e.g., Iloquinoxel); Or antibody therapy (e.g., GSK2857916) that targets BCMA; Or an antibody therapy (e.g., tbalumab) that targets BAFF or BLyss. In addition, the use of the antibodies and the antibodies of the present invention in compositions can be combined with a number of bisphosphonates (e.g., pamidronate, zoledronic acid). It has previously been described that APRIL protects MM cells from IL-6 deprivation, dexamethasone and bortezomib therapy (Moreaux et al, 2004, Blood 103 (8): 3148-57; Li et al., 2010, Med Oncol 27: 439-45). hAPRIL.01A has been shown to reverse the APRIL mediated survival of MM cells in the treatment of lanaridomide and dexamethasone (Tai et al., 2014, ASH poster 2098). In view of such findings in the art, the antibodies of the present invention are particularly useful for the treatment and / or prevention of corticosteroids such as dexamethasone, prednisolone, preferably dexamethasone, or thalidomide analogs, such as thalidomide, May be combined with additional therapeutic agents selected from tamarind, fomalidomide, especially lanalidomide, or bortezomide.

Diagnosis

APRIL detection in serum and / or tissues of a human subject is important because APRIL represents a disease, such as, but not limited to, an important marker for autoimmune diseases, inflammatory diseases and malignant tumors. For diagnostic applications, the antibody will typically be labeled (directly or indirectly) with a detectable moiety. Many labels are generally available that can be grouped into the following categories: biotin, fluorochrome, radioactive nucleotides, enzymes, iodine, and biosynthetic labels.

Soluble APRIL present in serum and other body fluids and / or tissues in different patient ranges appeared to be associated with the severity of the patient's disease. For example, chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma, NHL and multiple myeloma (MM), DLBCL patient (NHL), colorectal cancer, SLE, Patients suffering from atopic dermatitis and psoriatic arthritis, including Alzheimer's syndrome, psoriatic arthritis, multiple myelitis and ankylosing spondylitis, and patients with atopic dermatitis have dramatically increased serum levels of soluble APRIL. In addition, serum APRIL levels are elevated in patients suffering from IgA nephropathy (McCarthy et al., 2011, J. Clin. Invest . 121 (10): 3991-4002). In addition, serum APRIL levels are elevated in sepsis and serum APRIL levels predict death in severely ill patients (Jonsson et al., 1986, Scand J Rheumatol Suppl 61, 166-9; Roschke et al., 2002, J Immunol 169, 4314-21). Based on the proven binding properties of hAPRIL.01A, the antibodies of the invention can be used as a diagnostic tool to detect soluble APRIL in body fluids and / or tissues.

The antibodies of the present invention can be used in any known analytical method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies, A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The antibodies of the present invention can also be used for in vivo diagnostic assays. Generally, an antibody is labeled with a radionuclide such that the antigen or cell expressing it is localized using immunostimulatory or positron emission tomography.

Non-therapeutic use

According to another aspect of the invention, the antibody has other non-therapeutic uses. Non-therapeutic applications for the antibodies of the invention include flow cytometry, Western blotting, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry.

The antibody of the present invention can be used as an affinity purification reagent for protein A-Sepharose column, for example, through immobilization.

General Definition

The term "antibody" refers to any form of antibody that exhibits the desired biological activity, for example, by inhibiting binding of a ligand to its receptor or inhibiting ligand-induced signal transduction of a receptor. In the context of the present invention, the biological activity includes blocking the binding of APRIL to its receptor BCMA and / or TACI. Thus, the term "antibody" is used in its broadest sense and specifically includes, for example, the Duobody (TM) technology (Genmab) or Hexabody (TM) (Including full-length monoclonal antibodies) and multispecific antibodies (e. G., Bispecific antibodies). ≪ / RTI >

"Antibody fragment" and "antibody binding fragment" refer to an antigen-binding fragment of an antibody, typically comprising at least a portion of an antigen binding or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least a portion of the binding specificity of the parent antibody. Typically, the antibody fragment retains at least 10% of the parent binding activity, where such activity is expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; Diabody; Linear antibodies; Single chain antibody molecules, such as sc-Fv, unibody (description from Gen-map); Nanobody (technology from Ablynx); Domain antibody (technology from Domantis); And multispecific antibodies formed from antibody fragments. Engineered antibody variants are described in Holliger and Hudson, 2005, Nat. Biotechnol. 23: 1126-1136.

A "Fab fragment" is composed of CH1 and a variable region of one light chain and one heavy chain. The heavy chain of a Fab molecule can not form a disulfide bond with another heavy chain molecule.

The "Fc" region contains two heavy chain fragments comprising the C _H 1 and C _H 2 domains of the antibody. The two heavy chain fragments are held together by hydrophobic interaction of two or more disulphide bonds and C _H 3 domains.

"Fab disulfide bonds between the chains between the" fragments "is one light chain, and the V _H domain and contains the C _H 1 domain, F (ab ') to form _two molecules _of two Fab' 2 heavy chain fragments Lt; RTI ID = 0.0 _> of C _{< /} RTI >

Quot; F (ab ') ₂ fragment "refers to two light chains containing two light chains and a portion of the constant region between the C _H 1 and C _H 2 domains to form a chain interdisulfide bond between the two heavy chains . Thus the F (ab ') ₂ fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

"Fv region" includes variable regions from both heavy and light chains, but lacks constant regions.

A "single chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the V _H and V _L domains of an antibody, wherein such domains are within a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V _H domain and the V _L domain such that the scFv can form a desired structure for antigen binding. For review of scFv, see Pluckthun, 1994, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also International Patent Application Publication No. WO 88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.

"Diabody" is a small antibody fragment with two antigen-binding sites. Such fragments comprise a heavy chain variable domain (V _H ) linked to a light chain variable domain (V _L ) in the same polypeptide chain (V _H -V _L or V _L -V _H ). By using linkers that are too short to allow pairing between two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and two antigen-binding sites are created. Diabodies are described, for example, in EP 404,097; WO 93/11161; And Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448.

"Duobody" is a bispecific antibody with a normal IgG structure (Labrijn et al., 2013, Proc. Natl. Acad. Sci. USA 110 (13): 5145-5150).

"Hexabody" is an antibody with increased killing ability with regular structure and specificity (Diebolder et al., 2014, Science 343 (6176): 1260-3).

A "domain antibody fragment" is an immunologically functional immunoglobulin fragment that contains only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more V _H regions are covalently linked to the peptide linker to produce a divalent domain antibody fragment. The two V _H regions of the divalent domain antibody fragment can target the same or different antigens.

As used herein, the antibody hAPRIL.01A is a mouse antibody wherein the heavy chain has the amino acid sequence of SEQ ID NO: 55 and the light chain has the amino acid sequence of SEQ ID NO: 56.

For example, when at least one of the hinge cysteines normally involved in the disulfide bond between the heavy chains is altered in a known manner available to the skilled artisan, the antibody fragment of the present invention is capable of dimerization of the heavy chain with reduced disulfide binding capacity Lt; RTI ID = 0.0 > and / or < / RTI > In yet another embodiment, an antibody fragment, e. G., A fragment comprising an Fc region, is present in an intact antibody, wherein the biological function normally associated with the Fc region, such as FcRn binding, antibody half- Cell cytotoxicity) function, and / or complement binding (e. G., When the antibody has an ADCC function or a glycosylation profile required for complement binding).

The term "chimeric" antibody is intended to mean that a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) Or an antibody that belongs to another antibody class or subclass, as well as antibodies that are identical or homologous to corresponding sequences in fragments of such antibodies, as long as they exhibit the desired biological activity (see, for example, U.S. Patent No. 4,816,567, Morrison et al., 1984, Proc Natl Acad Sci USA 81: 6851-6855).

As used herein, the term "humanized antibody" refers to an antibody form containing a sequence from a non-human (e.g., murine) antibody, as well as a human antibody. Such antibodies contain minimal sequences derived from non-human immunoglobulins. In general, a humanized antibody will comprise at least one, typically two, variable domains in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all FR regions are of the human immunoglobulin sequence As shown in FIG. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. An essentially humanized form of rodent antibody will comprise the same CDR sequence as the parent rodent antibody, but may include specific amino acid substitutions for increased affinity, increased stability of the humanized antibody, or for other reasons.

The antibodies of the present invention also include antibodies in which the Fc region has been modified (or blocked) to provide a modified effector function. See, for example, U.S. Patent No. 5,624,821; WO 2003/086310; WO2005 / 120571; WO2006 / 0057702; Presta, 2006, Adv. Drug Delivery Rev. 58: 640-656. Such modifications can be used to enhance or inhibit various responses of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations in the Fc region include amino acid changes (substitution, deletion and insertion), glycosylation or deglycosylation, and addition of multiple Fc. Changes to Fc can also alter the antibody half-life in the therapeutic antibody, and a longer half-life will result in less frequent dosing, accompanied by increased convenience and decreased use of the material. Presta, 2005, J. Allergy Clin. Immunol. 116: 731, pp. 734-35.

The antibodies of the present invention also include antibodies of the non-compromised Fc region, for example isotype IgG1, which provide full effector function, which is complement-dependent cytotoxic (CDC) in the cells associated with the target for the antibody, Or antibody-dependent cellular cytotoxicity (ADCC).

Antibodies may be conjugated (e. G., Covalently linked) to molecules that improve antibody stability during storage or increase antibody half-life in vivo. Examples of molecules that increase half-life are albumin (e. G., Human serum albumin) and polyethylene glycol (PEG). Albumin-linked and PEGylated derivatives of antibodies can be prepared using techniques well known in the art. See, for example, Chapman, 2002, Adv. Drug Deliv. Rev. 54: 531-545; [Anderson and Tomasi, 1988, J. Immunol. Methods 109: 37-42; [Suzuki et al., 1984, Biochim. Biophys. Acta 788: 248-255; And [Brekke and Sandlie, 2003, Nature Rev. 2: 52-62.

The term "hypervariable region" as used herein refers to an amino acid residue of an antibody responsible for antigen-binding. The hypervariable region includes amino acid residues from "complementarity determining regions" or "CDRs " defined by sequence alignment, such as residues 24-34 (LI), 50-56 (L3) and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain (Rabat et al., 1991, Sequences of proteins of Immunological Interest, 5th Ed. (HVL) as defined herein, and / or as structurally defined, for example, light chain variable domains such as residues 26-32 (see LI ), 50-52 (L2) and 91-96 (L3) and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Leskl, 1987, J Mol. Biol. 196: 901-917).

"Framework" or "FR" residues or sequences are those variable domain residues or sequences other than the CDR residues as defined herein.

An antibody of the invention according to a particular embodiment may be an isolated antibody. An "isolated" antibody is one that identifies, isolates, isolates, or recovers its natural environment components. Contaminant components of its natural environment are substances that interfere with diagnostic or therapeutic use in antibodies and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody comprises (1) at least 95%, most preferably at least 99% by weight of the antibody as measured by the Lowry method, (2) To an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using coumadine blue or, preferably, silver staining will be. The isolated antibody is the antibody of the natural environment. Lt; RTI ID = 0.0 > recombinant < / RTI > intracellular antibody because one component will not be present. Generally, however, the isolated antibody may be produced by at least one purification step.

An "isolated" nucleic acid molecule is a nucleic acid molecule identified and separated from at least one contaminating nucleic acid molecule, which is generally associated with the natural source of the antibody nucleic acid. Isolated nucleic acid molecules are different from those found in nature. The isolated nucleic acid molecule is thus distinguishable from that present in the natural cell from the nucleic acid molecule. However, the isolated nucleic acid molecule includes, for example, a nucleic acid molecule contained within a cell that normally expresses an antibody whose nucleic acid molecule is at a chromosomal location different from that of the native cell.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i. E. Individual antibodies forming a population, except for possible naturally occurring mutations that may be present in minor amounts Are the same. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The phrase "monoclonal" refers to a characteristic of an antibody that is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring antibody production by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be prepared by the hybridoma method first described in Kohler et al., 1975, Nature 256: 495, or may be prepared by recombinant DNA methods (See, for example, U.S. Patent No. 4,816,567). Clackson et al., 1991, Nature 352: 624-628 and Marks et al., 1991, J. Mol. Biol . 222: 581-597], for example, a "monoclonal antibody" may be isolated from a phage antibody library using the techniques described. Monoclonal antibodies herein specifically include "chimeric" antibodies.

As used herein, the term "immune cell" includes cells that are hematopoietic and play a role in the immune response. Immune cells include lymphocytes such as B cells and T cells, natural killer cells, bone marrow cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

As used herein, "immunoconjugate" refers to a therapeutic moiety, for example, an anti-human APRIL antibody conjugated to a bacterial toxin, cytotoxic drug or radiation toxin, or fragment thereof. The toxin moiety may be conjugated to an antibody of the invention using methods available in the art.

As used herein, a sequence "variant" or "variant sequence" refers to a sequence that differs from the sequence listed at one or more amino acid residues while maintaining the biological activity of the parent molecule. The invention encompasses variants of the antibodies that are expressly described by various sequences. For the V _H domains CDR1, CDR2 and CDR3 sequences, according to some embodiments, the variant sequences comprise a total of six or more amino acid substitutions relative to the CDR1, CDR2 and CDR3 sequences, for example 1, 2, 3, 4, 5 Or 6 amino acid substitutions. Similarly, for the V _L domains CDR1, CDR2 and CDR3 sequences, according to some embodiments, the variant sequences comprise a total of six or fewer amino acid substitutions relative to the CDR1, CDR2 and CDR3 sequences, for example, 1, 2, 3, 4, 5 or 6 amino acid substitutions.

"Conservatively modified variants" or "conservative amino acid substitutions" refer to amino acid substitutions that are known to those skilled in the relevant art and which can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art will recognize that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, e.g., Watson, et al., Molecular Biology of the Gene , The Benjamin / Cummings Pub. Co., p. 224 (4th Edition 1987)). Preferably, such exemplary substitutions are made as described in Table 2 above.

As used herein, the term "about" refers to a value that is within an acceptable range of tolerances for a particular value, as determined by one of ordinary skill in the art, and is dependent on the manner in which the value is measured or determined, As shown in FIG. For example, "about" may mean at least one standard deviation per run in the art. Alternatively, "about," or "essentially comprising" may mean a range of up to 20%. Also, particularly with respect to biological systems or processes, such terms may mean less than or equal to 10 times the value. Where a specific value is provided in the applications and claims, unless otherwise stated, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable range of tolerances for this particular value.

The term "plurality" should be understood to mean one or more. Depending on the context of its use, the "plurality" may represent any suitable number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, "Multiple" may have the meaning of "plurality" in accordance with a particular embodiment. "Multiple" refers to any suitable number selected from 2, 3, 4, 5, 6, 7, 8, 9, 10 depending on the context of its use.

Quot; specifically binds " refers to a binding response that is crucial to the presence of a protein, e. G., APRIL, in a heterologous population of proteins and / or other biologics when presenting a ligand / receptor, antibody / do. Thus, under specified conditions, specific ligand / antigen binding to a particular receptor / antibody does not bind in significant amounts to other proteins present in the sample.

&Quot; Administration ", "therapy ", and" treatment ", when applied to an animal, a human, an experimental subject, a cell, a tissue, an organ or a biological fluid, Refers to the contact of an exogenous pharmaceutical, therapeutic, diagnostic agent or composition. &Quot; Administration ", "therapy ", and" treatment "can refer, for example, to therapy, pharmacodynamics, diagnosis, research and experimental methods. Treatment of the cells includes contact of the reagents to the cells, as well as contact of the reagents to the fluid when the fluid is in contact with the cells. &Quot; Administration ", "therapy" and "treatment" also refer to in vitro and in vitro treatment of cells, for example, by reagents, diagnostics, The terms "in vitro" and "in vitro" within the present description of the present invention may be used interchangeably with similar meanings.

Antibody DNA may also be obtained by replacing the coding sequence for the human heavy and light chain constant domains in place of the homologous murine sequences (US Pat. No. 4,816,567; Morrison, et al., 1984, Proc. Natl Acad. : 6851]), or by covalently linking all or part of a coding sequence for a non-immunoglobulin material (e.g., a protein domain) to an immunoglobulin coding sequence. Typically, such a non-immunoglobulin substance is an antigen-binding site that has a single antigen-binding site that is specific for an antigen and has a specificity for a different antigen, either by substituting the constant domain of the antibody, or by substituting the variable domain of one antigen- A chimera comprising another antigen-binding site produces antibodies.

Amino acid sequence variants of the anti-human APRIL antibodies of the present invention are prepared by introducing appropriate nucleotide changes into the coding DNA or by peptide synthesis. Such variants include, for example, deletion from, and / or insertion into, and / or substitution of, an indicated amino acid sequence for an anti-APRIL antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct as long as the final construct possesses the desired feature. Amino acid changes can also alter the post-translational processing of anti-APRIL antibodies, and can, for example, change the number or position of the glycosylation sites.

A useful method for identifying specific residues or regions of an anti-APRIL antibody polypeptide, which is a preferred location for mutagenesis, is referred to as "alanine scanning mutagenesis" as described in Cunningham and Wells, 1989, Science 244: 1081-1085 Lt; / RTI > Here, a residue or group of target residues is identified (e.g., charged residues such as Arg, Asp, His, Lys, and GIu), neutral or negatively charged amino acids (most preferably alanine or poly Alanine), which affects the interaction of amino acids with APRIL antigens. Amino acid residues that demonstrate functional susceptibility to substitution are then purified at the replacement site or by introducing additional or other variants to the replacement site. Thus, although the site for introducing the amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, Ala scanning or random mutagenesis is performed in the target codon or region to analyze the performance of the mutation at a defined site and screened for the desired activity in a variant of the expressed anti-APRIL antibody.

Normally, the amino acid sequence variant of the anti-APRIL antibody is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 75%, more preferably at least 80% Will have an amino acid sequence having at least 95%, 98% or 99% amino acid sequence similarity. The similarity or homology with respect to this sequence is as defined above.

Antibodies with the properties identified herein as desired can be screened for increased biological activity in vitro or for appropriate binding affinity. In order to screen for antibodies that bind to the same epitope of human APRIL as hAPRIL.01A, the routine cross-reactions described in, for example, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) Blocking analysis can be performed. Antibodies that bind to the same epitope appear to be cross-blocking in this assay, but cross-blocking can result from steric hindrance of antibody binding by antibody binding in an overlapping epitope, or even in a nearby non-overlapping epitope, The cross-blocking antibody will not necessarily bind precisely to the same epitope.

Alternatively, for example, see Champe et al., 1995, J. Biol. Chem. 270: 1388-1394) can be performed to determine whether an antibody binds to an epitope of interest. Quot; alanine scanning mutagenesis "as described in Cunningham and Wells, 1989, Science 244: 1081-1085, or point mutagenesis of amino acid residues in human APRIL. Some other forms also exist for the anti-APRIL antibodies of the invention Can be used to measure functional epitopes.

Another method for mapping epitopes of antibodies is described by Slootstra et al. (1996, MoI. Diversity 1: 87-96) and Timmerman et al. (Timmerman et al., 2007, J The present invention is to study the binding of antibodies to synthetic linear and CLIPS peptides that can be screened using a credit-card format mini PEPSCAN card as described in J. Mol. Recognit . 20: 283-299. Binding of the antibody to each peptide is measured in a PEPSCAN-based enzyme-linked immunoassay (ELISA).

Additional antibody binding to the same epitope as hAPRIL.01A can be achieved, for example, by screening raised antibodies against APRIL for binding to the epitope, or by screening peptides and fragments of human APRIL comprising epitope sequences ≪ / RTI > Antibodies that bind to the same functional epitope can be expected to exhibit similar biological activities, e. G., Similar APRIL binding and BCMA and TACI blocking activity, and such activity can be ascertained by functional analysis of the antibody.

The antibody may be selected from any class of immunoglobulins including IgM, IgG, IgD, IgA, and IgE. Preferably, the antibody is an IgG antibody. Any isotype of IgG, including IgGl, IgG2, IgG3, and IgG4, may be used. Variants of the IgG isotype are also expected. Antibodies can include sequences from one or more classifications or isotypes. Optimization of the requisite constant domain sequence to generate the desired biological activity is actually achieved by screening the antibody in the biological assays described in the Examples.

Thus, the classification of light chains can be used in the compositions and methods herein. Specifically, kappa, lambda, or variants thereof are useful in the compositions and methods of the present invention.

Antibodies and antibody fragments of the invention are also cytotoxic payload, for example, a cytotoxic agent or radiation nucleotides, ^{e.g., 99 Tc, 90 Y, 111} In, 32 P, 14 C, 125 I, 3 H, ^{131 I, 11 C, 15 O} , 13 N, 18 F, 35 S, 51 Cr, 57 To, 226 Ra, 60 Co, 59 Fe, 57 Se, 152 Eu, 67 Cu, 217 Ci, 211 At, 212 Pb , ⁴⁷ Sc, ¹⁰⁹ Pd, ²³⁴ Th, and ⁴⁰ K, ¹⁵⁷ Gd, ⁵⁵ Mn, ⁵² Tr, and ⁵⁶ Fe. Such antibody conjugates can be used in immunotherapy to selectively target and kill cells expressing the target (antigen to the antibody) on the surface thereof. Exemplary cytotoxic agents include lysine, vinca alkaloids, methotrexate, pseudomonas exotoxin, saponin, diphtheria toxin, cisplatin, doxorubicin, abritin toxin, gellonin and dobby antiviral proteins.

The antibodies and antibody fragments of the present invention may also be conjugated to a fluorescent moiety such as a rare earth chelate, fluorescein and derivatives thereof, rhodamine and derivatives thereof, isothiocyanate, fcoerythrin, picosan, alopecocaine, ophthalaldehyde, fluororescam, ¹⁵² Eu, dicylum, umbeliferone, luciferin, luminal label, isomeric label, aromatic acridinium ester label, imidazole label, acrylid salt label, oxalate ester label , Aquoline label, 2,3-dihydrophthalazinidione, biotin / avidin, a spin label, and a stable free radical.

Antibody molecules or protein molecules of the invention are described in Hunter et al., 1962, Nature 144: 945; David et al., 1974, Biochemistry 13: 1014); [Pain et al., 1981, J. Immunol. Meth . 40: 219]; And [Nygren, J., 1982, Histochem. and Cytochem . 30: 407], can be used. Methods of conjugating antibodies and proteins are conventional and well known in the art.

Antibody purification

When using recombinant techniques, the antibody can be produced in intracellular cytoplasmic space, or can be secreted directly into the medium. When the antibody is produced intracellularly, as a first step, particle debris, host cells or dissolved fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio / Technology 10: 163-167 describes the process of isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When the antibody is secreted into the medium, the supernatant from such an expression system is first concentrated, typically using an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor, such as PMSF, may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of contingent contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and affinity chromatography is preferred for purification techniques. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc region present in the antibody. Protein A can be used to purify antibodies based on human Ig. Gamma 1, Ig. Gamma 2, or Ig. Gamma 4 heavy chains (Lindmark et al., 1983, J. Immunol. Meth . 62: 1-13 ). Protein G is recommended for all mouse isotypes and human. Gamma. 3 (Guss et al., 1986, EMBO J 5: 1567-1575). Matrices to which affinity ligands are attached are mostly agarose, although other matrices are available.

Mechanically stable matrices, such as controlled void glass or poly (styrene divinyl) benzene, enable faster flow rates and shorter processing times than achieved by the agarose. Baker Bond ABX (TM) resin (JT Baker, Phillipsburg, NJ) is useful for purification when the antibody comprises a C _H 3 domain. Other techniques for protein purification include, for example, fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin, chromatography on anion or cation exchange resin (e. G., Polyaspartic acid column) Parous ™ chromatography, chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

In one embodiment, adsorption on a lectin substrate (e.g., a lectin affinity column) is used to purify the glycoprotein to remove the fucose-containing glycoprotein from the preparation and thus release the fucose-free glycoprotein It can be abundant.

Pharmaceutical formulation

The present invention includes pharmaceutical formulations of anti-human APRIL antibodies. To produce a pharmaceutical or bactericidal composition, the antibody, particularly the antibody or fragment thereof, may be mixed with a pharmaceutically acceptable carrier or excipient and may be prepared, for example, as described in Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984). Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions (see, for example, Hardman, Gennaro, 2000, Remington: The Science and Practice of Pharmacology , Lippincott, Williams, and Wilkins, New York, NY, et al., 2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics , McGraw-Hill, Pharmaceutical Dosage Forms: Parenteral Medications , Marcel Dekker, NY]; [Lieberman, et al. (Eds.), 1990. Tablets , Marcel Dekker, NY]; [Lieberman, et al (eds.), 1990, Pharmaceutical Dosage Forms:. Disperse Systems, Marcel Dekker, NY]; [Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

For example, to determine LD ₅₀ (lethal adult dose for 50% of the population) and ED ₅₀ (therapeutically effective dose at 50% of the population), either alone or in combination with another agent, The toxicity and therapeutic efficacy of the administered binding compounds, particularly antibodies, compositions, can be determined in cell cultures or in experimental animals by standard pharmaceutical procedures. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio between LD ₅₀ and ED ₅₀ . Data obtained from such cell culture assays and animal studies can be used to formulate a wide range of dosages for use in humans. Preferably, the dosage of such compounds is within a wide range of blood concentrations, including the ED ₅₀ , which has little or no toxicity. Dosages may vary within this range depending on the dosage form employed or the route of administration utilized.

Suitable routes of administration include parenteral administration, for example, intramuscular, intravenous or subcutaneous administration, and oral administration. Administration of the antibody for use in pharmaceutical compositions or for use in shampooing the methods of the invention may be by any of a variety of conventional routes such as oral ingestion, inhalation, topical application, or subcutaneous, intraperitoneal, parenteral, May be performed by intravenous injection. In one embodiment, the binding compounds of the invention are administered intravenously. In another embodiment, the binding compound of the invention is administered subcutaneously.

Alternatively, the antibody can be administered via direct injection of the antibody or antibodies into the site of action, in a local rather than systemic manner, e.g., often in a depot or sustained release formulation. In addition, the antibody may be administered in a targeted drug delivery system.

Guidance in selecting suitable doses of antibodies, cytokines and small molecules is available (see, for example, Wawrzynczak, 1996, Antibody Therapy , Bios Scientific Pub. ), 1991, Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY];. [Bach (ed), 1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY]; [Baert, et al., 1999, New Engl. J. Med. 341: 1966-1973; Slamon, et al., 2003, New Engl., J. Med 348: 601-608; .. 2001, New Engl J. Med 344: 783-792]; [Beniaminovitz, et al, 2000, New Engl J. Med 342:.... 613-619]; [Ghosh, et al, 2003, New Engl J. Med . 348: 24-32]; [Lipsky, et al., 2000, New Engl., J. Med 343: 1594-1602].

For example, a determination of a suitable dose is made by the clinician using parameters or factors that are known in the art for affecting treatment, or that are presumed, or predicted to affect, or affect treatment. Generally, the dose starts with an amount that is somewhat less than the optimal dose, and is then increased by a small amount of increment until a desired or optimal effect is achieved with respect to any negative side effects. Critical diagnostic scales include, for example, the symptoms of inflammation or a measure of the level of inflammatory cytokines produced.

A preferred dose protocol is one that involves a maximum dose or dose frequency to avoid significant undesirable side effects. The total weekly dose is generally at least 0.05 g / kg body weight, more usually at least 0.2 g / kg, most usually at least 0.5 g / kg, typically at least 1 g / kg, more typically at least 10 g / kg, most typically at least 100 μg / kg, preferably at least 0.2 mg / kg, more preferably at least 1.0 mg / kg, most preferably at least 2.0 mg / kg, optimally at least 10 mg / , More optimally at least 25 mg / kg, most optimally at least 50 mg / kg (see, for example, Yang, et al., 2003, New Engl. J. Med . 349: 427-434; [Liu, et al., 1999, J. Neurol Neurosurg Psych 67: 451-456] [Portielji, et al., 2002, New Engl , J. Med. et al., 2003, Cancer Immunol. Immunother . 52: 133-144). The desired dose of a small molecule therapeutic agent, e. G., A peptide mimetic, natural product, or organic chemical, is about the same as for an antibody or polypeptide on a mol / kg basis.

As used herein, "inhibiting" or "treating" or "treatment" refers to delaying the onset of symptoms associated with a disease and / or the progression of such symptoms And a reduction in severity. These terms further include improving existing symptoms, preventing further symptoms, and improving or preventing the underlying cause of such symptoms. Thus, this term indicates that beneficial results have been conferred in the vertebrate subject suffering from the disease.

An antibody of the invention for therapeutic purposes is administered in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" or "effective amount ", alone or in combination with additional therapeutic agents, refers to an amount of the antibody or composition effective to prevent or ameliorate a disease or condition to be treated when administered to a cell, Refers to the amount of antibodies. A therapeutically effective dose further refers to an amount of a compound sufficient to result in an improvement in the symptoms, for example, the treatment, healing, prevention or amelioration of the relevant medical condition, or the treatment, healing, prevention or amelioration of such a condition, do. When applied to the individual active ingredients administered alone, the therapeutically effective dose refers to these alone. When applied to a combination, the therapeutically effective dose refers to the combined amount of active ingredients that, when administered in combination, sequentially or concurrently, results in a therapeutic effect. An effective amount of therapeutic agent is typically at least 10%; Generally at least 20%; Preferably by at least about 30%; More preferably by at least 40%, and most preferably by at least 50%.

Methods for co-administration or therapy with a second therapeutic agent are well known in the art and are described, for example, in Hardman, et al. (Eds.), 2001, Goodman and Gilman ' s Pharmacological Basis of Therapeutics , ed., McGraw-Hill, New York, NY]; [Poole and Peterson (eds.), 2001, Pharmacotherapeutics for Advanced Practice: A Practical Approach , Lippincott, Williams & Wilkins, Phil. [Chabner and Longo (eds.), 2001, Cancer Chemotherapy and Biotherapy , Lippincott, Williams & Wilkins, Phila.

The pharmaceutical compositions of the present invention may also be used in combination with cytotoxic agents, chemotherapeutic agents, cell proliferation inhibitors, angiogenesis inhibitors or antimetabolites, tumor targets, immunostimulants, immunomodulators or cytotoxic agents, cell proliferation inhibitors, But are not limited to, antibodies conjugated to the antibody. The pharmaceutical composition may also be used in conjunction with other therapeutic modalities, such as surgery, chemotherapy and radiation.

The present invention will now be further described and supported with reference to the following non-limiting experiments.

Experiment

Experiment 1

Anti-APRIL humanized antibody design

CDR grafting

A unique antibody, hAPRIL.01A (WO2010 / 100056), which binds to human APRIL was first identified. Mouse hAPRIL.01A antibody is humanized with CDR-grafting techniques (see, for example, US Pat. No. 5,225, 539 and Williams, DG et al., 2010, Antibody Engineering, volume 1, Chapter 21) . First, a strategy to identify human germline sequences using IgBLAST (Ye J. et al., 2013, Nucleic Acids Res. 41: W34-40) is designed. In hAPRIL.01A VH, the human germline sequence IGKV1-16 * 01 (65.3% identity) was identified in the human germline sequence IGHV1-3 * 01 (70.4% identity), and hAPRIL.01A VL.

Then, an IMGT database (Publication 201222-4: 161905, Index 04-Jun-2012) (Lefranc, M.-P. et al., 1999, Nucleic Acids Res. 27: 209- 212) was constructed with a database containing all human maturation sequences available. These sequences were queried using TBLASTN (2.2.26 +) to identify template sequences that demonstrated the highest identity to the hAPRIL.01A VH and VL sequences (SEQ ID NOS: 3 and 4, respectively). CDR2 and CDR3 (SEQ ID NOS: 8 to 10), respectively, or a similar, preferably identical CDR (SEQ ID NOS: 5 to 7) and VL CDR1, CDR2 Three VH and seven VL sequences were chosen to represent the length.

In the heavy chain, the framework encoded by GenBank (Benson, D.A. et al., 2013, Nucleic Acids Res. 41 (D1): D36-42) under accession numbers AF022000, AB363149, and AB063827 hAPRIL.01A VH CDRs, which resulted in the following cDNA constructs: SEQ ID NOs: 11, 13 and 15. For light chains, GenBank accession numbers AX375917, DD272023, AB363267, AJ241396, DI152527 , And the framework encoded by DQ840975 was selected for the linear grafting of the hAPRIL.01A VL CDR, resulting in the following cDNA constructs: SEQ ID NOS: 19, 21, 23, 25, 27 and 29.

Based on the common sequence from alignment of the 25 best match sequences (E-values 5e-46 through 9e-43) from the TBLASTN results, additional heavy chain sequences were designed, resulting in the following cDNA constructs: SEQ ID NO: 17 .

To determine the structural effect of humanization of framework residues, a homology model of hAPRIL.01A antibody was generated using WHATIF (Krieger E. et al., 2003, Methods Biochem Anal. 44: 509-23). Template for VH and VL chains, 2GKI (Kim YR et al., 2006, J. Biol. Chem. 281: 15287-15295) and 2AEQ (Venkatramani L. et al., 2006, J. Mol. -663) were each transfected with a BLASTP study (Altschul, SF et < RTI ID = 0.0 > al., 1990, J. Mol. Biol. 215: 403-410). The VH and VL chains could be combined as Fab fragments using the MUSTANG alignment (Konagurthu A.S. et al., 2006, Proteins 64: 559-574), which was guided by the 2AEQ template. An established homology model of hAPRIL.01A was used to select residues that were affected by humanization and which could affect the functionality of humanized constructs and were evaluated whether or not selected residues were replaced: Six VL templates ( VL15), it was determined to replace VL residues Y49 and Y87 with smaller S49 and F87.

Identification of signal peptides

A human germline repertoire matching the mouse hAPRIL.01A VH and VL was identified using NCBI IgBlast (BLASTN) (Ye J. et al., 2013, Nucleic Acids Res 41 (Web Server issue): W34-40) It was used to select secretory leaders for VH and VL: VL based on gonadal IGHV1-3 * 01 (NCBI registration number X62107), and VL based on gonadal IGKV16 * 01 (NCBI registration number X62109). All humanized VH and VL constructs (SEQ ID NO: 58) and the VL secretory leader sequence "MDMRVLAQLLGLLLLCFPGARC" (SEQ ID NO: 60) coded by SEQ ID NO: 57 were used to generate the VH secretory leader sequence "MDWTWRILFLVAAATGAHS" Lt; / RTI >

An IgG4 version of the humanized antibody was prepared with a stabilized Adair mutant (Angal S. et al., 1993, Mol Immunol. 30: 105-108), wherein serine 241 (designated Rabat) .

Experiment 2

Synthesis, subcloning, expression, binding

synthesis

The cDNAs encoding the humanized VH and VL constructs, codons 11, 13, 15, 17, 21, 23, 25, 27 and 29 were codon optimized using OptGene software (version 2.0.6.0) It was chemically synthesized with Clear (BaseClear). The sequence was then cloned into pUC57 vector (base clear) using 5'-Hindlll and 3'-Apal (VH) or 3'-BsiWI (VL) restriction endonuclease cleavage sites.

Subcloning

Using the above-mentioned restriction endonuclease cleavage site, pcDNA3.1 (+) containing the human IgG4 constant domain (CH1-CH3, GenBank Accession No. K01316) cloned into the EcoRI and Hindlll restriction endonuclease cleavage sites, ) Vector (Invitrogen). ≪ / RTI > (+) Vector containing the human CL (kappa) domain (GenBank accession number J00241) cloned into Hindlll and EcoRI restriction endonuclease cleavage sites using the above-mentioned restriction endonuclease cleavage site RTI ID = 0.0 > (Invitrogen). ≪ / RTI > According to the manufacturer's instructions, constructs were transformed into subcloning efficient DH5 [alpha] competent cells (Invitrogen). Plasmid DNA was isolated using a Qiagen Plasmid Midi Kit (QIAGEN) according to the manufacturer's protocol. The integrity of the construct was confirmed by DNA sequencing (Macrogen).

Expression and binding

The plasmids encoding the VH and VL constructs were mixed in a 1: 3 ratio (4 ug in total) and were transfected using the Lipofectamine 2000 transfection reagent (Invitrogen) Were transiently expressed by transfection into embryonic kidney cells (HEK293T / 17, ATCC-CRL-11268). Cell supernatants were harvested after 5 days and assayed for antibody expression and binding to APRIL using enzyme-linked immunoassay (ELISA). In these ELISAs, all incubation steps followed a washing step with PBST (PBS containing 0.01% Tween 20). Maxisorb 96-well plates (Nunc) were coated with 0.5 μg / ml anti-FLAG (Sigma) or anti-IgG4 (Jackson laboratories) Lt; / RTI > Subsequently, anti-FLAG coated 96-well plates were incubated with FLAG-tagged human APRIL at room temperature for 1 hour. The supernatant and its dilutions were then incubated for 1 hour and then incubated with mouse anti-human IgG HRP-conjugate (Southern Biotechnology) for 1 hour.

Immunoreactivity was visualized with 100 [mu] l of TMB Stabilized Chromagen (Invitrogen). The reaction was stopped with 100 μl of 0.5 MH ₂ SO ₄ and the absorbance was measured at 450 and 620 nM.

Experiment 3

Purification and stability

refine

A subset of the humanized antibodies described above was selected for further analysis. Again, the plasmids encoding the VH and VL constructs were mixed in a 1: 3 ratio (32 [mu] g) and (8 * 10 < ⁶ >) using lipofectamine 2000 transfection reagent (Invitrogen) And transiently expressed by transfection into HEK293T human embryonic kidney cells (HEK293T). The supernatant was collected (10 ml) and the antibody was excised using MabSelect Sure Protein A resin according to the manufacturer's instructions (GE Healthcare). The buffer was replaced for PBS using a Zeba desalting column (Thermo Scientific). The concentration of the purified antibody was measured based on OD280 (Nanodrop ND-1000). The binding of the purified antibody to APRIL was established using the APRIL ELISA described above. The blocking ability of humanized antibodies by BCMA and TACI receptors was tested in a competitive ELISA. In these ELISAs, all incubation steps followed washing steps with PBST (PBS containing 0.01% Tween 20). Maxisov 96-well plates (Nuke) were coated with 0.5 μg / ml Fc-BCMA (R & D Systems) or Fc-TACI (R & D Systems) and incubated overnight at 4 ° C. FLAG-tagged APRIL and pre-mixed humanized antibody and dilutions thereof were incubated for 1 hour and then incubated with anti-FLAG HRP-conjugate (Sigma) for 1 hour. Immunoreactivity was visualized with 100 占 퐇 of TMB-stabilized chromagens (Invitrogen). The reaction was stopped with 100 μl of 0.5 MH ₂ SO ₄ and the absorbance was measured at 450 and 620 nM. The calculated EC ₅₀ and IC _50, which represent the concentration at which 50% of the total binding signal or block is observed, are shown in Table 5 .

Table 5: Combination of EC50 values, APRIL. Blocking of APRIL binding to IC50, BCMA-Fc. C4-hAPRIL.01A was used as an experimental reference in each ELISA. nc indicated inhibition, but IC50 could not be calculated due to inappropriate fitting.

A similar blocking effect was observed in blocking the APRIL binding to TACI-Fc.

Surprisingly, the combination of VL10-VL14 and VH11-VH14 did not exhibit nano- or micromolar EC50 values, only a combination of the VL15 framework sequence and the selected VH framework sequence resulted in antibodies with functional APRIL binding properties.

stability

To determine the effect of humanization on stabilization of antibodies, humanized antibodies were exposed to various temperature ranges for 10 minutes. The purified antibody was diluted with 3.16 [mu] g / ml and its dilution in PBS. These solutions were then exposed to either 65 캜 or 70 캜 and residual binding was measured after heat treatment of the antibody using FLAG-tagged APRIL ELISA assay as previously described (see Table 6).

Table 6: Residual binding of (humanized) antibodies to FLAG-APRIL as measured by ELISA. Binding is measured at three concentrations: 3.16, 1 and 0.316 [mu] g / ml. The% binding at 65 or 70 ° C is expressed as the% binding observed for each antibody at room temperature (= 100%).

Experiment 4

Improvement of binding, blocking and stability by back mutation and vernier residues

Improvement of binding and blocking by white mutation

Sequence and structural levels were analyzed to understand the molecular basis for differences in binding and blocking of different VH / VL combinations. A homology model of humanized antibody was made as described above. The template selected for both VH and VL was 3HC4 (Jordan J. L. et al., 2009, Proteins 77: 832-841). Based on careful analysis of the generated model, the inventors of the present invention have assumed that residue S72 in the selected VH chain is important for the orientation of the CDR2 loop. To investigate this hypothesis, the mutant R72S was introduced into VH14, resulting in VH14_1, SEQ ID NO: 32, encoded by the nucleotide sequence of SEQ ID NO: 31. Antibody 14_1.15 was tested for binding and blocking as described above. As shown in Table 7, the binding and blocking of antibody 14_1.15 is improved relative to the binding of antibody 14.15 as shown in Table 5.

Table 7: Blocking of APRIL binding and APRIL binding to BCMA-Fc of antibody 14_1.15. hAPRIL.01A and C4-hAPRIL.01A were used as experimental references in each ELISA.

Similar IC50 values were obtained in the blocking of APRIL binding to TACI-Fc.

Vernier residue

Sequence and structural level analyzes were performed to further improve the binding and blocking of antibody 14_1.15. A homology model of this hAPRIL.01A analog was prepared as described above. The selected template for the VH chain was 2GKI and 4GMT in the VL chain (Lee P.S. et al., 2012, PNAS 109: 17040-17045) and assembled as Fab fragments guided by template 2AEQ.

Since residues close to the CDR can affect loop deformation, they have been studied in detail. In the analysis, the inventor identified a number of potentially related vernier residues (Foote J. et al., 1992, J. Mol. Biol. 224: 487-499). To evaluate their relevance, they were replaced by mouse amino acids.

The introduction of the mutant M70I resulted in VH14_1C (SEQ ID NOS: 33 and 34), the mutation T74K was present in VH14_1D (SEQ ID NOS: 35 and 36) and the mutant Q1E caused VH14_1E (SEQ ID NOS: 37 and 38). The combined mutation of R67K and V68A resulted in VH 14_1G, SEQ ID NO: 39, Antibodies were tested for binding, blocking, and stability as described above. As shown in Table 8, surprisingly, the binding and blocking are improved two to three times. In particular the mutations introduced into the antibody VH14_1G.VL15 show surprisingly significant improvement.

Table 8: Combination and blocking of antibody 14_1.15 and verniform domain mutations. hAPRIL.01A was used as a reference in each ELISA.

In addition, the stability of the substituted humanized antibodies was improved as determined using a thermal stability study as described in Example 3 (see Table 9).

Table 9 : Residual binding of (humanized) antibody to FLAG-APRIL as measured by ELISA. The binding is measured at three concentrations: 3.16, 1 and 0.316 [mu] g / ml. The% bond at 65 or 70 ° C is expressed as the% bond observed for each of the antibodies at room temperature (= 100%).

Experiment 5

14 1G.15 proves to be more efficient in in vivo suppression.

To demonstrate the in vivo blocking effect of APRIL analogue antibodies on APRIL function, we tested the ability of the antibody to block the NP-phole-induced humoral response in mice. The mice used were both APRIL transgenic (TG) mice and asymptomatic (WT) rats aged 8-10 weeks for the C57BL / 6 background. APRIL transgenic mice express human APRIL under the Lck-terminal promoter, which directs transgene expression to mature thymocytes and peripheral T lymphocytes (Stein et al., 2002, J Clin Invest 109, 1587-98). Mice were raised in animal facilities of university medical institutions, and experiments were approved by the Institutional Ethics Committee. The mice were divided into several groups and treated as follows: WT mice were treated with PBS (200 μl) and the APRIL transgenic mouse aged groups were treated with the following molecules: hAPRIL.01A or 14 1G.15 And 200 [mu] g / mouse in 200 [mu] g PBS on day 3) or PBS. At day 0, mice were immunized with NP-Picol (0 day; 100 쨉 ip with 250 ug of immunogen). Blood was collected via the tail vein on days -1, 3, 7, and 10. The anti- (4-hydroxy-nitrophenacetyl) (NP) -specific antibodies (IgM, IgG and IgAa / 2) were analyzed by ELISA using diluted sera as described above (Hardenberg et al. Immunol Cell Biol, 86 (6): 530-4, (2008); Guadagnoli et al., 2011, Blood 117 (25): 6856-65). Briefly, 96-well ELISA plates (Greiner) were coated with 5 ug / ml NP-BSA (Biosearch Technologies) in sodium carbonate buffer (pH 9.6) overnight at 4 캜. Wells were blocked with 1% BSA at 37 < 0 > C for 1 hour and incubated with diluted serum for 2 hours at room temperature. HRP-conjugated isotype-specific antibodies (goat anti-mouse IgG, IgA and IgM, from Southern Biotech). All dilutions were made in PBS / BSA 1% / Tween 20 0.05%. As evident from Figure 1, both hAPRIL.01A and 14_1G.15 inhibited T-cell dependent B-cell responses in vivo. hAPRIL.01A inhibited this response less effectively than 14_1G.15. PBS and mouse IgG1 as isotype-matched controls did not affect IgA, IgM and IgG anti-NP responses.

SEQUENCE LISTING <110> Aduro Biotech Holdings, Europe B.V. <120> Altered APRIL binding antibodies <130> WO2016 / 110587 <140> PCT / EP2016 / 050314 <141> 2016-01-08 <150> NL 2014108 <151> 2014-01-09 <160> 60 <170> BiSSAP 1.2 <210> 1 <211> 363 <212> DNA <213> Mus musculus <220> <221> source <222> 1..363 <223> / mol_type = "unassigned DNA"       / organism = "Mus musculus" <400> 1 gaggtccagt tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggata cacattcact agctatgtga tgcactgggt gaagcagaag 120 cctgggcagg gccttgagtg gattggatat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcaa ggccacagtg acttcagaca agtcctccgg cacagcctac 240 atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aaggggcttg 300 ggttacgccc tttactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 360 tca 363 <210> 2 <211> 321 <212> DNA <213> Mus musculus <220> <221> source <222> 1..321 <223> / mol_type = "unassigned DNA"       / organism = "Mus musculus" <400> 2 gacattgtga tgacccagtc tcaaaaattc aagtccacat cagtaggaga cagggtcagc 60 gtcacctgca aggccagtca gaatgtgggt aataatgtag cctggtatca acagaaagca 120 gggcaatctc ctaaagcact gatttcctcg gcatccaacc gtgacagtgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240 gaagacttgg cagactattt ctgtcagcaa tataacatct atccattcac gttcggctcg 300 gggacaaagt tggaaataaa a 321 <210> 3 <211> 121 <212> PRT <213> Mus musculus <400> 3 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Val Thr Ser Asp Lys Ser Ser Gly Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser         115 120 <210> 4 <211> 107 <212> PRT <213> Mus musculus <400> 4 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Lys Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn             20 25 30 Val Ala Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Lys Ala Leu Ile         35 40 45 Ser Ser Ala Ser Asn Arg Asp Ser Gly Val Pro Asp Arg Phe Thr Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ile Tyr Pro Phe                 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 5 <211> 8 <212> PRT <213> Mus musculus <400> 5 Gly Tyr Thr Phe Thr Ser Tyr Val 1 5 <210> 6 <211> 16 <212> PRT <213> Mus musculus <400> 6 Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 <210> 7 <211> 12 <212> PRT <213> Mus musculus <400> 7 Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 8 <211> 11 <212> PRT <213> Mus musculus <400> 8 Lys Ala Ser Gln Asn Val Gly Asn Asn Val Ala 1 5 10 <210> 9 <211> 7 <212> PRT <213> Mus musculus <400> 9 Ser Ala Ser Asn Arg Asp Ser 1 5 <210> 10 <211> 10 <212> PRT <213> Mus musculus <400> 10 Gln Gln Tyr Asn Ile Tyr Pro Phe Thr Phe 1 5 10 <210> 11 <211> 363 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..363 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VH sequence"       / organism = "Artificial Sequence" <400> 11 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg cttctggata cacattcact agctatgtga tgcattgggt gcgccaggcc 120 cccggacaaa ggcttgagtg gatgggatat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag agtcaccatt accagggaca catccgcgag cacagcctac 240 atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc gagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccaag ggaccacggt caccgtctcc 360 tca 363 <210> 12 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VH sequence <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 13 <211> 363 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..363 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VH sequence"       / organism = "Artificial Sequence" <400> 13 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata cacattcact agctatgtga tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240 atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccaag ggaccacggt caccgtctcg 360 agc 363 <210> 14 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VH sequence <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 15 <211> 363 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..363 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VH sequence"       / organism = "Artificial Sequence" <400> 15 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata cacattcact agctatgtga tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccaag ggacaatggt caccgtctcg 360 agc 363 <210> 16 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VH sequence <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser         115 120 <210> 17 <211> 363 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..363 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VH sequence"       / organism = "Artificial Sequence" <400> 17 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ccggcgccag cgtgaaggtg 60 agctgcaagg ccagcggata cacattcact agctatgtga tgcactgggt gagacaggcc 120 cccggccagg gcctggagtg gatgggctat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag agtgaccatg accagagaca ccagcgccag caccgcctac 240 atggagctga gcagcctgag aagcgacgac accgccgtgt actactgcgc cagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccagg gcaccaccgt gaccgtgagc 360 agc 363 <210> 18 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VH sequence <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 19 <211> 321 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..321 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VL sequence"       / organism = "Artificial Sequence" <400> 19 gacattgtga tgacccagtc tcaaaaattc atgtccacat ccgtaggaga cagggtcagc 60 atcacctgca aggccagtca gaatgtgggt aataatgtag cctggtatca acagaaacca 120 ggacaatctc ctaaattgct gatttactcg gcatccaacc gtgacagtgg agtccctgat 180 cgcttctcag gcagtgggtc tgggacagat ttcactctca ccatcagcaa tatgcagtct 240 gaagacctgg cagattattt ctgccagcaa tataacatct atccattcac gttcggaggg 300 gggaccaagc tggaaatcaa a 321 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VL sequence <400> 20 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Asn Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ile Tyr Pro Phe                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 21 <211> 321 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..321 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VL sequence"       / organism = "Artificial Sequence" <400> 21 gacattgtga tgacccagtc tcaaaaattc atgcccacat cagtaggaga cagggtcagc 60 gtcacctgca aggccagtca gaatgtgggt aataatgtag cctggtatca acagaaacca 120 gggcaatctc ctaaagcact gatttactcg gcatccaacc gtgacagtgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcaccaa tgtgcagtct 240 gaagacttgg cagagtattt ctgtcagcaa tataacatct atccattcac gttcggtgct 300 gggaccaagc tggagctgaa a 321 <210> 22 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VL sequence <400> 22 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Pro Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile         35 40 45 Tyr Ser Ala Ser Asn Arg Asp Ser Gly Val Pro Asp Arg Phe Thr Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ile Tyr Pro Phe                 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys             100 105 <210> 23 <211> 321 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..321 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VL sequence"       / organism = "Artificial Sequence" <400> 23 gaaattgtgt tgacgcagtc tccttccacc cagtctgcat ctgtaggaga cagagtcacc 60 atcacttgca aggccagtca gaatgtgggt aataatgtag cctggtatca gcagaaacca 120 gggaaagccc ctaaactcct aatctattcg gcatccaacc gtgacagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagag ttcactctca ccatcagcag cctgcagcct 240 gatgattttg caacttatta ctgccagcaa tataacatct atccattcac gtttggccag 300 gggaccaagc tggagatcaa a 321 <210> 24 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VL sequence <400> 24 Glu Ile Val Leu Thr Gln Ser Ser Ser Thr Gln Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Asn Arg Asp Ser Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Phe                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 25 <211> 321 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..321 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VL sequence"       / organism = "Artificial Sequence" <400> 25 gacatcgtga tgacccagtc tccttctacc ctgtctgcat ctgtgggaga cagagtcacc 60 atcacttgca aggccagtca gaatgtgggt aataatgtag cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctattcg gcatccaacc gtgacagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagag ttcactctca ccatcagcag cctgcagcct 240 gatgattttg caacttatta ctgccagcaa tataacatct atccattcac gtttggccag 300 gggaccaagc tggagatcaa a 321 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VL sequence <400> 26 Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Asn Arg Asp Ser Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Phe                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 27 <211> 321 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..321 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VL sequence"       / organism = "Artificial Sequence" <400> 27 gatatcctga tgacccagtc tcaaaaaatc atgcccacat cagtgggaga cagggtcagc 60 gtcacctgca aggccagtca gaatgtgggt aataatgtag cctggtatca acagaaacca 120 ggacagtctc ctaaagcact gatttactcg gcatccaacc gtgacagtgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcaccaa tgtgcagtct 240 gaggacttgg cagagtattt ctgtcagcaa tataacatct atccattcac gttcggtgct 300 gggaccaagc tggacctgaa a 321 <210> 28 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VL sequence <400> 28 Asp Ile Leu Met Thr Gln Ser Gln Lys Ile Met Pro Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile         35 40 45 Tyr Ser Ala Ser Asn Arg Asp Ser Gly Val Pro Asp Arg Phe Thr Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ile Tyr Pro Phe                 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Asp Leu Lys             100 105 <210> 29 <211> 321 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..321 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VL sequence"       / organism = "Artificial Sequence" <400> 29 gacatcgtga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgca aggccagtca gaatgtgggt aataatgtag cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctcttcg gcatccaacc gtgacagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagag ttcactctca ccatcagcag cctgcagcct 240 gatgattttg caacttattt ctgccagcaa tataacatct atccattcac gtttggccag 300 gggaccaagc tggagatcaa a 321 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VL sequence <400> 30 Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Ser Ser Ala Ser Asn Arg Asp Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Asn Ile Tyr Pro Phe                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 31 <211> 363 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..363 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VH sequence"       / organism = "Artificial Sequence" <400> 31 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ccggcgccag cgtgaaggtg 60 agctgcaagg ccagcggata cacattcact agctatgtga tgcactgggt gagacaggcc 120 cccggccagg gcctggagtg gatgggctat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag agtgaccatg accagtgaca ccagcgccag caccgcctac 240 atggagctga gcagcctgag aagcgacgac accgccgtgt actactgcgc cagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccagg gcaccaccgt gaccgtgagc 360 agc 363 <210> 32 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VH sequence <400> 32 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 33 <211> 363 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..363 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VH sequence"       / organism = "Artificial Sequence" <400> 33 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ccggcgccag cgtgaaggtg 60 agctgcaagg ccagcggata cacattcact agctatgtga tgcactgggt gagacaggcc 120 cccggccagg gcctggagtg gatgggctat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag agtgaccatc accagtgaca ccagcgccag caccgcctac 240 atggagctga gcagcctgag aagcgacgac accgccgtgt actactgcgc cagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccagg gcaccaccgt gaccgtgagc 360 agc 363 <210> 34 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VH sequence <400> 34 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Ile Thr Ser Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 35 <211> 363 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..363 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VH sequence"       / organism = "Artificial Sequence" <400> 35 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ccggcgccag cgtgaaggtg 60 agctgcaagg ccagcggata cacattcact agctatgtga tgcactgggt gagacaggcc 120 cccggccagg gcctggagtg gatgggctat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag agtgaccatg accagtgaca agagcgccag caccgcctac 240 atggagctga gcagcctgag aagcgacgac accgccgtgt actactgcgc cagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccagg gcaccaccgt gaccgtgagc 360 agc 363 <210> 36 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VH sequence <400> 36 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Met Thr Ser Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 37 <211> 363 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..363 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VH sequence"       / organism = "Artificial Sequence" <400> 37 gaggtccagc ttgtgcagtc tggggctgag gtgaagaagc ccggcgccag cgtgaaggtg 60 agctgcaagg ccagcggata cacattcact agctatgtga tgcactgggt gagacaggcc 120 cccggccagg gcctggagtg gatgggctat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag agtgaccatg accagtgaca ccagcgccag caccgcctac 240 atggagctga gcagcctgag aagcgacgac accgccgtgt actactgcgc cagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccagg gcaccaccgt gaccgtgagc 360 agc 363 <210> 38 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VH sequence <400> 38 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 39 <211> 363 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..363 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin VH sequence"       / organism = "Artificial Sequence" <400> 39 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ccggcgccag cgtgaaggtg 60 agctgcaagg ccagcggata cacattcact agctatgtga tgcactgggt gagacaggcc 120 cccggccagg gcctggagtg gatgggctat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcaa agcgaccatg accagtgaca ccagcgccag caccgcctac 240 atggagctga gcagcctgag aagcgacgac accgccgtgt actactgcgc cagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccagg gcaccaccgt gaccgtgagc 360 agc 363 <210> 40 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin VH sequence <400> 40 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Met Thr Ser Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 41 <211> 1344 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..1344 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin heavy chain sequence"       / organism = "Artificial Sequence" <400> 41 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg cttctggata cacattcact agctatgtga tgcattgggt gcgccaggcc 120 cccggacaaa ggcttgagtg gatgggatat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag agtcaccatt accagggaca catccgcgag cacagcctac 240 atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc gagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccaag ggaccacggt caccgtctcc 360 tcagcatcca ccaagggccc atccgtcttc cccctggcgc cctgctccag gagcacctcc 420 gagagcacag ccgccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacgaag 600 acctacacct gcaacgtaga tcacaagccc agcaacacca aggtggacaa gagagttgag 660 tccaaatatg gtcccccatg cccaccatgc ccagcacctg agttcctggg gggaccatca 720 gtcttcctgt tccccccaaa acccaaggac actctcatga tctcccggac ccctgaggtc 780 acgtgcgtgg tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg 840 gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac 960 aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccagga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 tccgacggct ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag 1260 gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 1320 agcctctccc tgtctctggg taaa 1344 <210> 42 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin heavy chain sequence <400> 42 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly     210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg                 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro             260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala         275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val     290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr                 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu             340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys         355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser     370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser                 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala             420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys         435 440 445 <210> 43 <211> 1344 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..1344 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin heavy chain sequence"       / organism = "Artificial Sequence" <400> 43 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata cacattcact agctatgtga tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240 atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccaag ggaccacggt caccgtctcg 360 agcgcatcca ccaagggccc atccgtcttc cccctggcgc cctgctccag gagcacctcc 420 gagagcacag ccgccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacgaag 600 acctacacct gcaacgtaga tcacaagccc agcaacacca aggtggacaa gagagttgag 660 tccaaatatg gtcccccatg cccaccatgc ccagcacctg agttcctggg gggaccatca 720 gtcttcctgt tccccccaaa acccaaggac actctcatga tctcccggac ccctgaggtc 780 acgtgcgtgg tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg 840 gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac 960 aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccagga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 tccgacggct ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag 1260 gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 1320 agcctctccc tgtctctggg taaa 1344 <210> 44 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin heavy chain sequence <400> 44 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly     210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg                 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro             260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala         275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val     290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr                 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu             340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys         355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser     370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser                 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala             420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys         435 440 445 <210> 45 <211> 1344 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..1344 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin heavy chain sequence"       / organism = "Artificial Sequence" <400> 45 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata cacattcact agctatgtga tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccaag ggacaatggt caccgtctcg 360 agcgcatcca ccaagggccc atccgtcttc cccctggcgc cctgctccag gagcacctcc 420 gagagcacag ccgccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacgaag 600 acctacacct gcaacgtaga tcacaagccc agcaacacca aggtggacaa gagagttgag 660 tccaaatatg gtcccccatg cccaccatgc ccagcacctg agttcctggg gggaccatca 720 gtcttcctgt tccccccaaa acccaaggac actctcatga tctcccggac ccctgaggtc 780 acgtgcgtgg tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg 840 gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac 960 aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccagga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 tccgacggct ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag 1260 gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 1320 agcctctccc tgtctctggg taaa 1344 <210> 46 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin heavy chain sequence <400> 46 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly     210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg                 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro             260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala         275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val     290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr                 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu             340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys         355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser     370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser                 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala             420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys         435 440 445 <210> 47 <211> 1344 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..1344 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin heavy chain sequence"       / organism = "Artificial Sequence" <400> 47 caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ccggcgccag cgtgaaggtg 60 agctgcaagg ccagcggata cacattcact agctatgtga tgcactgggt gagacaggcc 120 cccggccagg gcctggagtg gatgggctat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcag agtgaccatg accagagaca ccagcgccag caccgcctac 240 atggagctga gcagcctgag aagcgacgac accgccgtgt actactgcgc cagaggcttg 300 ggttacgccc tttactatgc tatggactac tggggccagg gcaccaccgt gaccgtgagc 360 agcgcatcca ccaagggccc atccgtcttc cccctggcgc cctgctccag gagcacctcc 420 gagagcacag ccgccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacgaag 600 acctacacct gcaacgtaga tcacaagccc agcaacacca aggtggacaa gagagttgag 660 tccaaatatg gtcccccatg cccaccatgc ccagcacctg agttcctggg gggaccatca 720 gtcttcctgt tccccccaaa acccaaggac actctcatga tctcccggac ccctgaggtc 780 acgtgcgtgg tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg 840 gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac 960 aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccagga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 tccgacggct ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag 1260 gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 1320 agcctctccc tgtctctggg taaa 1344 <210> 48 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin heavy chain sequence <400> 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly     210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg                 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro             260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala         275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val     290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr                 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu             340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys         355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser     370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser                 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala             420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys         435 440 445 <210> 49 <211> 642 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1.642 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin light chain sequence"       / organism = "Artificial Sequence" <400> 49 gacattgtga tgacccagtc tccttccact ctctctgcat ctgtgggaga cagagtaacc 60 atcacttgca aggccagtca gaatgtgggt aataatgtag cctggtatca gcagaaacca 120 gggaaagccc ctaagctact gatctcttcc gcatccaacc gggacagtgg tgtgccctca 180 aggtttagcg gcagtggatc agggacagag ttcacattga ccatatccag cctgcagcct 240 gatgattttg ctacttattt ctgccaacaa tataacattt acccattcac gtttggccag 300 ggcaccaagc tagagatcaa acggacggtt gctgcaccct ctgtctttat cttcccgcca 360 tctgatgaac agttgaagtc cggaacagcc tctgttgtgt gcctgctgaa taacttttat 420 ccccgcgagg cgaaagttca gtggaaggtg gataacgccc tccaatcagg caattcccag 480 gagagtgtga cagagcaaga ttccaaggac tcaacctaca gcctcagcag tactttaact 540 ctgagcaaag cagactacga gaagcacaaa gtctacgctt gcgaagtcac ccatcagggc 600 cttagctcgc ccgtcacaaa gagctttaac aggggagaat gt 642 <210> 50 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin light chain sequence <400> 50 Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Ser Ser Ala Ser Asn Arg Asp Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Asn Ile Tyr Pro Phe                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 51 <211> 1344 <212> DNA <213> Artificial Sequence <220> <221> source <222> 1..1344 <223> / mol_type = "unassigned DNA"       / note = "Engineered immunoglobulin heavy chain sequence"       / organism = "Artificial Sequence" <400> 51 caggtccaac ttgtgcagtc tggggctgaa gtgaagaagc ccggcgctag tgtgaaggtt 60 tcatgtaagg cttctggata cacatttact tcatatgtaa tgcactgggt gcgtcaagcc 120 cctggccagg gcctggagtg gatggggtat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggaaa agcaactatg accagtgata ctagcgcttc aaccgcctac 240 atggagctga gcagcttaag aagcgacgac accgccgtgt actattgtgc caggggcttg 300 ggttacgccc tttattatgc tatggactac tggggtcagg gcaccacagt gaccgttagc 360 tctgcatcta ctaagggacc atccgtcttc cccctggcgc catgctcccg cagtacaagt 420 gagagcacag cagccctggg ctgtttggta aaggactact tccccgaacc tgtgactgtg 480 tcttggaact caggcgccct gactagcggc gtgcacactt tccctgctgt cctacagtcc 540 tcaggactat actccctctc gtctgtggtg acagtgcctt cctcatcatt gggaacgaaa 600 acctatactt gcaacgttga tcacaagccc agcaacacca aggtggacaa gagagttgag 660 tccaaatatg gtcccccatg tccaccatgt ccagcacctg agtttcttgg cggaccaagt 720 gttttcctgt tccccccaaa acccaaggat actctcatga taagtcgcac ccctgaagtc 780 acttgcgtgg tggtggacgt tagccaggaa gatcccgaag tccaattcaa ctggtacgta 840 gatggcgtag aagtgcataa tgcgaagaca aagccgagag aggagcagtt taattcgacg 900 tatcgggtgg tcagcgtcct cacagtcctg caccaggact ggctgaacgg caaggagtat 960 aagtgcaagg tctccaacaa aggtctcccg tcctccattg agaaaacaat ctccaaagca 1020 aaagggcagc cccgagaacc acaagtgtac accctgcccc catctcagga ggagatgacc 1080 aagaaccagg tcagtcttac ctgcctggtc aaaggctttt atccctcaga tatcgccgtt 1140 gagtgggaaa gcaatgggca gccggagaac aactacaaga ccacgcctcc cgttctggat 1200 tctgacggat cgttcttttt atacagcagg ctaaccgtgg acaagtctcg gtggcaggaa 1260 gggaatgtat tttcttgcag tgtaatgcat gaggctctgc acaatcatta cacacagaag 1320 tctctctccc tgtctcttgg taaa 1344 <210> 52 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Engineered immunoglobulin heavy chain sequence <400> 52 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Met Thr Ser Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly     210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg                 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro             260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala         275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val     290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr                 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu             340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys         355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser     370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser                 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala             420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys         435 440 445 <210> 53 <211> 1335 <212> DNA <213> Mus musculus <220> <221> source <222> 1.1335 <223> / mol_type = "unassigned DNA"       / organism = "Mus musculus" <400> 53 gaggtccagt tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggata cacattcact agctatgtga tgcactgggt gaagcagaag 120 cctgggcagg gccttgagtg gattggatat attaatcctt ataatgatgc tcctaaatac 180 aatgagaagt tcaaaggcaa ggccacagtg acttcagaca agtcctccgg cacagcctac 240 atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aaggggcttg 300 ggttacgccc tttactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 360 tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 420 aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 480 acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctggagtct 540 gacctctaca ctctgagcag ctcagtgact gtcccctcca gccctcggcc cagcgagacc 600 gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 660 agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 720 ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 780 gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 840 gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca 900 gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg 960 gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga 1020 ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa 1080 gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag 1140 tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgaa cacgaatggc 1200 tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact 1260 ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc 1320 cactctcctg gtaaa 1335 <210> 54 <211> 642 <212> DNA <213> Mus musculus <220> <221> source <222> 1.642 <223> / mol_type = "unassigned DNA"       / organism = "Mus musculus" <400> 54 gacattgtga tgacccagtc tcaaaaattc aagtccacat cagtaggaga cagggtcagc 60 gtcacctgca aggccagtca gaatgtgggt aataatgtag cctggtatca acagaaagca 120 gggcaatctc ctaaagcact gatttcctcg gcatccaacc gtgacagtgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240 gaagacttgg cagactattt ctgtcagcaa tataacatct atccattcac gttcggctcg 300 gggacaaagt tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 360 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 420 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 480 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 540 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 600 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gt 642 <210> 55 <211> 445 <212> PRT <213> Mus musculus <400> 55 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Ala Pro Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Val Thr Ser Asp Lys Ser Ser Gly Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Gly Tyr Ala Leu Tyr Tyr Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Ser Ser         115 120 125 Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val     130 135 140 Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Glu Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro             180 185 190 Ser Ser Pro Arg Ser Ser Glu Thr Val Thr Cys Asn Val Ala His Pro         195 200 205 Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly     210 215 220 Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys                 245 250 255 Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln             260 265 270 Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln         275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu     290 295 300 Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg 305 310 315 320 Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys                 325 330 335 Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro             340 345 350 Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr         355 360 365 Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln     370 375 380 Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn Gly 385 390 395 400 Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu                 405 410 415 Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn             420 425 430 His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys         435 440 445 <210> 56 <211> 214 <212> PRT <213> Mus musculus <400> 56 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Lys Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn             20 25 30 Val Ala Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Lys Ala Leu Ile         35 40 45 Ser Ser Ala Ser Asn Arg Asp Ser Gly Val Pro Asp Arg Phe Thr Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ile Tyr Pro Phe                 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala             100 105 110 Pro Thr Val Ser Ile Phe Pro Ser Ser Glu Gln Leu Thr Ser Gly         115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile     130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser                 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr             180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser         195 200 205 Phe Asn Arg Asn Glu Cys     210 <210> 57 <211> 57 <212> DNA <213> Homo sapiens <220> <221> source <222> 1..57 <223> / mol_type = "unassigned DNA"       / organism = "Homo sapiens" <400> 57 atggactgga cctggaggat cctctttttg gtggcagcag ccacaggtgc ccactcc 57 <210> 58 <211> 19 <212> PRT <213> Homo sapiens <400> 58 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser              <210> 59 <211> 66 <212> DNA <213> Homo sapiens <220> <221> source <222> 1..66 <223> / mol_type = "unassigned DNA"       / organism = "Homo sapiens" <400> 59 atggacatga gagtcctcgc tcagctcctg gggctcctgc tgctctgttt cccaggtgcc 60 agatgt 66 <210> 60 <211> 22 <212> PRT <213> Homo sapiens <400> 60 Met Asp Met Arg Val Leu Ala Gln Leu Leu Gly Leu Leu Leu Leu Cys 1 5 10 15 Phe Pro Gly Ala Arg Cys             20

Claims (22)

an antibody comprising an antibody fragment, e. g. an antibody fragment, having an antigen binding site of hAPRIL.01A, which is an APRIL-binding antibody comprising an antibody analog, e. g. antibody fragment, competing for binding to the same epitope of human APRIL , Wherein the APRIL-binding antibody comprises a plurality of antigen binding sites comprising V _H and V _L domains, wherein the framework sequence of the V _H domain in the antigen binding portion comprises SEQ ID NOs: 12, 14, 16, 18, Has at least 70% sequence similarity with the framework sequence of the V _H amino acid sequence selected from SEQ ID NO: 14 or 18, wherein the framework sequence of the V _L domain has at least 70% sequence similarity to the framework amino acid sequence of SEQ ID NO: 30 , APRIL-binding antibody. The method according to claim 1,
- at least one and preferably all three of CDR1, CDR2, CDR3 in said V _H domain are selected and / or selected from the group consisting of SEQ ID NOS: 5, 6, 7, or any variant of said sequence;
- at least one and preferably all three of CDR1, CDR2, CDR3 in said V _L domain are selected from the group consisting of SEQ ID NOs: 8, 9 and 10, or any variant of said sequence, respectively. 3. The polypeptide according to claim 1 or 2, wherein the V _H domain amino acid sequence is at least 70% sequence homologous to the amino acid sequence selected from SEQ ID NO: 42, 44, 46, 48, And wherein the V _L domain amino acid sequence is in a light chain amino acid sequence having at least 70% sequence similarity to the amino acid sequence of SEQ ID NO: 50. Article according to any one of the preceding claims, wherein the amino acid at position 72 S from the V _H amino acid sequences, the V _H amino acid sequence is preferably from SEQ ID NO: 32, 34, 36, 38 or 40 Antibody selected. 5. The antibody of any one of claims 1 to 4, wherein at least 70% sequence similarity is at least 85% sequence similarity. 5. The antibody of any one of claims 1 to 4, wherein at least 70% sequence similarity is at least 90% sequence similarity. 5. The antibody of any one of claims 1 to 4, wherein at least 70% sequence similarity is at least 95% sequence similarity. 5. The antibody of any one of claims 1 to 4, wherein at least 70% sequence similarity is at least 99% sequence similarity. 9. The method according to any one of claims 1 to 8, wherein the amino acid at position 67 in the V _H amino acid sequence is K, the amino acid at position 68 is A, and the V _H amino acid sequence is preferably SEQ ID NO: &Lt; / RTI &gt; 10. An antibody according to any one of claims 1 to 9, wherein the antibody has one or more of the following characteristics:
Binding to human APRIL with a K _D of about 100 nM or less;
Blocking the binding of human APRIL to human BCMA and human TACI with an IC ₅₀ of about 100 nM or less;
Block binding of hAPRIL.01A to human APRIL with an IC ₅₀ of about 100 nM or less. 10. An isolated polynucleotide encoding the V _H domain and / or the V _L domain of an antibody according to any one of claims 1 to 10 wherein the polynucleotide sequence encoding the V _H domain is preferably selected from the group consisting of SEQ ID NO: , 13, 15, 17, more preferably a polynucleotide sequence having at least 70% sequence similarity with a polynucleotide sequence selected from SEQ ID NO: 13 or 17, wherein the polynucleotide sequence encoding the V _L domain is preferably a polynucleotide sequence having at least 70% Polynucleotide sequence having at least 70% sequence similarity to a polynucleotide sequence selected from SEQ ID NO: 29. An expression unit comprising a plurality of expression vectors comprising a plurality of the polynucleotides of claim 11 under the control of suitable regulatory sequences, wherein the plurality of polynucleotides comprises a V _H of the antibody according to any one of claims 1 to 10 Domain and a V _L domain and wherein the polynucleotide sequence coding for the V _H domain is made in an expression vector that is the same as or different from the polynucleotide sequence coding for the V _L domain. 12. A host cell comprising a plurality of polynucleotides of claim 11 and / or an expression unit of claim 12, wherein the expression unit preferably comprises polynucleotide sequence coding for the V _H domain and polynucleotide sequence coding for the V _L domain And an expression vector comprising the same. 11. A method of producing an antibody according to any one of claims 1 to 10,
a) culturing the host cell according to claim 13 under culture conditions in which the plurality of polynucleotides are expressed in a culture medium to produce a polypeptide comprising the light chain variable region and the heavy chain variable region; And
b) recovering the polypeptide from the host cell or culture medium. Use of an antibody according to any one of claims 1 to 10 as a carrier or diluent, preferably a pharmaceutically acceptable carrier or diluent, and optionally a multiplicity of other active compounds, in particular melphalan, Corticosteroids such as corticosteroids such as dexamethasone, prednisolone, thalidomide analogs, such as corticosteroids, such as, for example, corticosteroids, Antitumor agents such as thalidomide, lanaridomide, fomalidomide, kinase inhibitors such as ivermitinib, idaralidis, CD20, such as rituximab, opatumum, ovinotoumum, Antibody therapy targeting CD52, such as Alemtuzumab, antibody therapy targeting CD38, such as daraturomat, antibodies targeting IL-6 or IL-6 receptors E. G., Antibody therapy that targets erythropoiemia, BCMA, e. G., GSK 2857916, BAFF, or BLyss, which target &lt; / RTI &gt; In combination with a plurality of therapeutically active compounds selected from antibody therapies such as tavanam, bisphosphonates such as pamidronate or zoledronic acid, or bortezomide. 10. Antibody according to any one of claims 1 to 10 for use in therapy directed against one or more selected from the following, preferably selected from the following a to e:
a. Inhibiting immune cell proliferation and / or survival;
b. Treatment of cancer;
c. Treatment of autoimmune diseases;
d. Treatment of inflammatory diseases; or
e. Treatment of conditions in which lowering of immunoglobulin levels is beneficial. A method of treating a subject, preferably a human subject, comprising administering a therapeutically effective amount of an antibody according to any one of claims 1 to 10. 18. The method of claim 17, wherein the treatment is aimed at one or more selected from:
a. Inhibiting immune cell proliferation and / or survival;
b. Treatment of cancer;
c. Treatment of autoimmune diseases;
d. Treatment of inflammatory diseases; or
e. Treatment of conditions in which lowering of immunoglobulin levels is beneficial. Diagnostic methods, such as, for example, in diagnostic methods selected from in vitro or in vitro diagnostic methods, for example, flow cytometry, Western blotting, enzyme linked immunosorbent assay (ELISA) or immunohistochemistry, &Lt; RTI ID = 0.0 &gt; 10. &Lt; / RTI &gt; A heavy chain variable region selected from the group consisting of VH11.VL15, VH12.VL15, VH13.VL15, VH14.VL15, VH14_1.VL15, VH14_1C.VL15, VH14_1D.VL15, VH14_1E.VL15, and VH14_1G.VL15. Containing humanized antibody. 20. A polynucleotide encoding a humanized antibody according to claim 20. An expression vector comprising a polynucleotide encoding a humanized antibody according to claim 20 operably linked to a regulatory sequence configured to provide expression of the humanized antibody.

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